Gene Set Overrepresentation Analysis Research Articles

Proteostasis as a fundamental principle of Tau immunotherapy.

The microtubule-associated protein Tau is a driver of neuronal dysfunction in Alzheimer's disease and other tauopathies. In this process, Tau initially undergoes subtle changes to its abundance, subcellular localisation and a vast array of post-translational modifications including phosphorylation, that progressively result in the protein's somatodendritic accumulation and dysregulation of multiple Tau-dependent cellular processes. Given the various loss- and gain-of-functions of Tau in disease and the brain-wide changes in the proteome that characterise tauopathies, we asked whether targeting Tau would restore the alterations in proteostasis observed in disease. Therefore, by phage display, we generated a novel pan-Tau antibody, RNJ1, that preferentially binds human Tau and neutralises proteopathic seeding activity in multiple cell lines, and benchmarked it against a clinically tested pan-Tau antibody, HJ8.5 (murine version of tilavonemab). We then evaluated both antibodies, alone and in combination, in the K3 tauopathy mouse model, showing reduced Tau pathology and improvements in neuronal function following 14 weekly treatments, without obtaining synergy for the combination. These effects were more pronounced in female mice. To investigate the molecular mechanisms contributing to improvements in neuronal function, we employed quantitative proteomics, phosphoproteomics and kinase prediction analysis to first establish alterations in K3 mice relative to WT controls at the proteome level. In female K3 mice, we found 342 differentially abundant proteins, which are predominantly involved in metabolic and microtubule-associated processes, strengthening previously reported findings of defects in several functional domains in multiple tauopathy models. We next asked whether antibody-mediated Tau target engagement indirectly affects levels of deregulated proteins in the K3 model. Importantly, both immunotherapies, in particular RNJ1, induced abundance shifts towards a restoration to wild-type levels (proteostasis). A total of 257 of 342 (∼75%) proteins altered in K3 were closer in abundance to WT levels after RNJ1 treatment, and 73% after HJ8.5 treatment. However, the magnitude of these changes was less pronounced than that observed with RNJ1, as reflected by a far smaller number of differentially abundant proteins. Furthermore, analysis of the phosphoproteome showed an even stronger restoration effect with RNJ1, with ∼82% of altered phosphopeptides in K3 showing a shift to WT levels, and 75% with HJ8.5. Gene set over-representation analysis (ORA) further confirmed that proteins undergoing restoration are involved in biological pathways affected in K3 mice. Together, our study suggests that a Tau immunotherapy-induced restoration of proteostasis links target engagement and treatment efficacy.

Network-medicine approach for the identification of genetic association of parathyroid adenoma with cardiovascular disease and type-2 diabetes.

Primary hyperparathyroidism is caused by solitary parathyroid adenomas (PTAs) in most cases (⁓85%), and it has been previously reported that PTAs are associated with cardiovascular disease (CVD) and type-2 diabetes (T2D). To understand the molecular basis of PTAs, we have investigated the genetic association amongst PTAs, CVD and T2D through an integrative network-based approach and observed a remarkable resemblance. The current study proposed to compare the PTAs-associated proteins with the overlapping proteins of CVD and T2D to determine the disease relationship. We constructed the protein-protein interaction network by integrating curated and experimentally validated interactions in humans. We found the $11$ highly clustered modules in the network, which contain a total of $13$ hub proteins (TP53, ESR1, EGFR, POTEF, MEN1, FLNA, CDKN2B, ACTB, CTNNB1, CAV1, MAPK1, G6PD and CCND1) that commonly co-exist in PTAs, CDV and T2D and reached to network's hierarchically modular organization. Additionally, we implemented a gene-set over-representation analysis over biological processes and pathways that helped to identify disease-associated pathways and prioritize target disease proteins. Moreover, we identified the respective drugs of these hub proteins. We built a bipartite network that helps decipher the drug-target interaction, highlighting the influential roles of these drugs on apparently unrelated targets and pathways. Targeting these hub proteins by using drug combinations or drug-repurposing approaches will improve the clinical conditions in comorbidity, enhance the potency of a few drugs and give a synergistic effect with better outcomes. This network-based analysis opens a new horizon for more personalized treatment and drug-repurposing opportunities to investigate new targets and multi-drug treatment and may be helpful in further analysis of the mechanisms underlying PTA and associated diseases.

EXPANSION: a webserver to explore the functional consequences of protein-coding alternative splice variants in cancer genomics.

EXPANSION (https://expansion.bioinfolab.sns.it/) is an integrated web-server to explore the functional consequences of protein-coding alternative splice variants. We combined information from Differentially Expressed (DE) protein-coding transcripts from cancer genomics, together with domain architecture, protein interaction network, and gene enrichment analysis to provide an easy-to-interpret view of the effects of protein-coding splice variants. We retrieved all the protein-coding Ensembl transcripts and mapped Interpro domains and post-translational modifications on canonical sequences to identify functionally relevant splicing events. We also retrieved isoform-specific protein-protein interactions and binding regions from IntAct to uncover isoform-specific functions via gene-set over-representation analysis. Through EXPANSION, users can analyze precalculated or user-inputted DE transcript datasets, to easily gain functional insights on any protein spliceform of interest. EXPANSION is freely available at http://expansion.bioinfolab.sns.it/. The code of the scripts used for EXPASION is available at: https://github.com/raimondilab/expansion. Datasets associated to this resource are available at the following URL: https://doi.org/10.5281/zenodo.8229120. The web-server was developed using Apache2 (https://https.apache.org/) and Flask (v2.0.2) (http://flask.pocoo.org/) for the web frontend and for the internal pipeline to handle back-end processes. We additionally used the following Python and JavaScript libraries at both back- and front-ends: D3 (v4), jQuery (v3.2.1), DataTables (v2.3.2), biopython (v1.79), gprofiler-officia l(v1.0.0), Mysql-connector-python (v8.0.31). To construct the API, Fast API library (v0.95.1) was used.

PS-BPB06-5: URINARY CELL TRANSCRIPTOMICS: A NON-INVASIVE EXPRESSION READOUT OF KIDNEY GENES OF RELEVANCE TO HYPERTENSION

Objective: Hypertension affects 35% of the global adult population and is the leading preventable risk factor for premature death globally. The kidney is the key organ of blood pressure regulation in the body. Here, we compare the gene expression profile from renal cells shed naturally into the urine and kidney tissue to determine if urine can provide a non-invasive readout of kidney gene expression and whether hypertension-associated genes can be quantified in urine. Design and Method: Urinary cell and kidney tissue samples were collected from 33 human participants and were both profiled by poly-A RNA-sequencing, generating an average of 30 million paired reads per sample. These were quantified using the standard Genotype Tissue Expression (GTEx) project pipeline and were compared against 43 different human tissues and other bodily fluids using transcriptomic correlation. RNA-sequencing quality metrics were calculated by RNA-SeQC software. Gene set overrepresentation analysis for Gene Ontology (GO) terms and Kyoto Encyclopaedia of Genes and Genomes (KEGG) employed Fishers exact test. Enriched and enhanced kidney genes were collected from the Human Protein Atlas (HPA). Results: Our RNA-sequencing metric analysis revealed that urinary cells can generate robust data of comparable or superior quality to that of saliva at similar read coverage. The top one hundred most highly expressed urinary cell genes show an enrichment for biological themes shared with the renal transcriptome, including immunity (P = 1.2x10–7), glucose metabolism (P = 1.3x10–5) and renal mineral reabsorption (P = 9.8x10–4). In an analysis all protein-coding genes across 43 human tissues/cell-types, urinary cells showed the highest level of transcriptomic correlation with kidney cortex (r2 = 0.65) and kidney medulla (r2 = 0.64). This correlation between urinary cells and kidney tissue was particularly strong (r2 = 0.72) in an analysis restricted to highly specific kidney genes (including uromodulin and the Na-K-Cl cotransporter NKCC2 [loop diuretic target]). 98% (176 out of 179) of kidney genes with a known causal association to blood pressure were expressed in urinary cells. Their urinary expression demonstrated strong correlation with their abundance in kidney cortex (r2 = 0.68) and medulla (r2 = 0.64). Conclusions: Standard poly-A RNA-sequencing of cells harvested from urinary sediments produces robust gene expression profiles. These profiles provide a non-invasive insight into transcriptome of the kidney and permit measuring expression of kidney genes of relevance to BP regulation and hypertension.

Metabolic Dysfunctions Driven By PRPF8 Explain Cases with Ring Sideroblasts without SF3B1 Mutations: Similarity/ Differences of Genotype/ Phenotype Association

SF3B1 mutations (SF3B1MT) mostly identify a group of MDS with ringed sideroblasts (RS), isolated erythroid dysplasia and favorable prognosis. Besides SF3B1, we and others have demonstrated that other genes might be involved in RS formation either due to convergent metabolic pathways or erythroid driver genes. MDS subtypes characterized by multilineage dysplasia lacking SF3B1MT and presenting with RS are paradigmatic examples of such a possibility. In addition, RS in AML is often unrecognized by hematopathologists due to the focus on the presence of blasts as a predominant feature and/ or the reduction of erythroid precursors inherent to blast infiltration. However, when we previously studied defects in PRPF81, a central but unappreciated target of somatic lesions in MDS, we found a correlation with the RS phenotype. To follow up on this initial finding, we compiled a well-annotated cohort of patients seen at the Cleveland Clinic to assess phenotype/ genotype association of PRPF8MT and RS. NGS results including PRPF8 mutational status of 3,545 cases with myeloid neoplasia were reviewed. Calls were retained after assessment of pathogenicity by the ACMG and VarSome classifiers. Excluding cases with concomitant SF3B1MT, we found PRPF8MT (median VAF 38% [3-79]) in 78 (2%) patients. In total, 83% of the mutations were missense, whereas 15% were truncating. Homology sequence between yeast and humans denoted that hotspots mapped to highly conserved amino acids. D1598 and C1594, localizing in the U6-snRNA interacting domain (1442-1601), were mutated in 14% and 5% of the patients, respectively. T418S mapped in a central domain of several SFs (PROCN) and was found in 9% of the cases, whereas the F1099fs alteration was detected in 5% of our cohort. Prussian blue stain showed the presence of RS in 25% of PRPF8MT. Since PRPF8 maps to the proximal regions of 17p, we also noticed that three informative cases carried somatic PRPF8MT and 17p11.2 deletion or loss of chr.17 (1/2), all showing the presence of RS. We thus screened a more complete cohort of chr.17 abnormalities (88% in the context of a complex karyotype) to investigate their contribution to RS formation. While 66% had RS, the high blast count may have concealed their presence in cases lacking this feature because of their predominant AML or high risk MDS. Notably, we also found 8 additional patients in whom SNP-A showed cryptic losses such as del17p11.2 and 17p13.1 (n=7) or UPD (n=2) containing elevated RS. In this cohort, TP53 was the most commonly mutated gene (49%) followed by ASXL1 and DNMT3A (10% each), BCOR/L1 and TET2 (7.5%). In regard to other SFMT, no patient had SRSF2MT, three had U2AF1S34/Q157, two carried somatic DDX41c.139-5C>T/A225D (with -5/del5q) and one patient had a LUC7L2G48* without -7/del7p. While the coincidental presence of marrow RS in TP53MT lacking SF3B1MT has been previously demonstrated2, no study has investigated whether RS might be a result of shared metabolic feedback mechanisms common to SFMT. We thus analyzed RNA splicing and gene expression (GE)3 of patients with single PRPF8MT (n=34) and SF3B1MT (n=281) finding overlapping splicing features in the a-hemoglobin synthesis HBA2 gene. Upregulation of HBA2 has been described in SF3B1MT patients4,5 to correlate with impaired erythrocyte maturation and its accumulation in early erythrocytes. Interestingly enough, given our data on chr.17 abnormalities, we also found that cases with single TP53MT (n=52) displayed high covariance features for HBA2, a possible culprit in cases carrying RS but unmutated for SF3B1 and PRPF8. Indeed by applying gene-set overrepresentation analysis to PRPF8MT and TP53MT GE profiles, we clustered the top 5 categories (FDR< .001) in erythrocytes differentiation (KLF1, EPB42), homeostasis and development (ALAS2, GATA1, RHAG, SLC4A1) suggesting that these genes might inversely correlate with HBA2 expression and be responsible for the formation of RS. Our study demonstrates that PRPF8MT is another molecular lesion phenocopying SF3B1MT via a compensatory metabolic mechanism responsible for the failure of terminal erythroid differentiation. In analogy, given the proximal genomic location of PRPF8 and TP53 we do not exclude that this commonality might be involved in TP53MT with RS as well. These likely phenocopying mechanisms may enable therapeutic opportunities (luspatercept) in cases lacking SF3B1MT presenting with RS and transfusion-dependent anemia.

Transcriptional profiles in the mouse amygdala after a cognitive judgment bias test largely depend on the genotype.

Background: The amygdala is crucial for emotional cognitive processing. Affective or emotional states can bias cognitive processes, including attention, memory, and decision-making. This can result in optimistic or pessimistic behaviors that are partially driven by the activation of the amygdala. The resulting emotional cognitive bias is a common feature of anxiety and mood disorders, both of which are interactively influenced by genetic and environmental factors. It is also known that emotional cognitive biases can be influenced by environmental factors. However, little is known about the effects of genetics and/or gene-environment interactions on emotional cognitive biases. We investigated the effects of the genetic background and environmental enrichment on the transcriptional profiles of the mouse amygdala following a well-established cognitive bias test. Methods: Twenty-four female C57BL/6J and B6D2F1N mice were housed either in standard (control) conditions or in an enriched environment. After appropriate training, the cognitive bias test was performed on 19 mice that satisfactorily completed the training scheme to assess their responses to ambiguous cues. This allowed us to calculate an "optimism score" for each mouse. Subsequently, we dissected the anterior and posterior portions of the amygdala to perform RNA-sequencing for differential expression and other statistical analyses. Results: In general, we found only minor changes in the amygdala's transcriptome associated with the levels of optimism in our mice. In contrast, we observed wide molecular effects of the genetic background in both housing environments. The C57BL/6J animals showed more transcriptional changes in response to enriched environments than the B6D2F1N mice. We also generally found more dysregulated genes in the posterior than in the anterior portion of the amygdala. Gene set overrepresentation analyses consistently implicated cellular metabolic responses and immune processes in the differences observed between mouse strains, while processes favoring neurogenesis and neurotransmission were implicated in the responses to environmental enrichment. In a correlation analysis, lipid metabolism in the anterior amygdala was suggested to influence the levels of optimism. Conclusions: Our observations underscore the importance of selecting appropriate animal models when performing molecular studies of affective conditions or emotional states, and suggest an important role of immune and stress responses in the genetic component of emotion regulation.

The impact of PrsA over-expression on the Bacillus subtilis transcriptome during fed-batch fermentation of alpha-amylase production.

The production of the alpha-amylase (AMY) enzyme in Bacillus subtilis at a high rate leads to the accumulation of unfolded AMY, which causes secretion stress. The over-expression of the PrsA chaperone aids enzyme folding and reduces stress. To identify affected pathways and potential mechanisms involved in the reduced growth, we analyzed the transcriptomic differences during fed-batch fermentation between a PrsA over-expressing strain and control in a time-series RNA-seq experiment. We observe transcription in 542 unannotated regions, of which 234 had significant changes in expression levels between the samples. Moreover, 1,791 protein-coding sequences, 80 non-coding genes, and 20 riboswitches overlapping UTR regions of coding genes had significant changes in expression. We identified putatively regulated biological processes via gene-set over-representation analysis of the differentially expressed genes; overall, the analysis suggests that the PrsA over-expression affects ATP biosynthesis activity, amino acid metabolism, and cell wall stability. The investigation of the protein interaction network points to a potential impact on cell motility signaling. We discuss the impact of these highlighted mechanisms for reducing secretion stress or detrimental aspects of PrsA over-expression during AMY production.

Identification of HOX signatures contributing to oral cancer phenotype

The role of evolutionarily conserved homeobox-containing HOX genes as transcriptional regulators in the developmental specification of organisms is well known. The contribution of HOX genes involvement in oral cancer phenotype has yet to be fully ascertained. TCGA-HNSC HTSeq-counts and clinical data were retrieved from the GDC portal for oral cavity neoplasms. GEO datasets (GSE72627, GSE30784, GSE37991) were accessed and analyzed using GEO2R. Differential HOX gene expression was profiled using the DESeq2 R package with a log2 fold change cut-off (− 1 and + 1) and Benjamini–Hochberg p-adjusted value at ≤ 0.01. Gene set over-representation analysis and semantic analysis associated with the disease ontology was performed using the ClusterProfiler R package, and pathway over-representation analysis was performed using IMPaLa. HOX protein interaction network was constructed using the Pathfind R package. HOX phenotype associations were performed using Mammalian Phenotype Ontology, Human Phenotype Ontology, PhenGenI associations, Jensen tissues, and OMIM entries. Drug connectivity mapping was carried out with Dr. Insight R package. HOXA2 was upregulated in oral dysplasia but silenced during tumor progression. Loss of HOXB2 expression was consistent in the potentially malignant oral lesions as well as in the primary tumor. HOXA7, HOXA10, HOXB7, HOXC6, HOXC10, HOXD10, and HOXD11 were consistently upregulated from premalignancy to malignancy and were notably associated with risk factors. Overrepresentation analysis suggested HOXA10 was involved in the transcriptional misregulation contributing to the oral cancer phenotype. HOX genes subnetwork analysis showed crucial interactions with cell cycle regulators, growth responsive elements, and proto-oncogenes. Phenotype associations specific to the oral region involving HOX genes provide intrinsic cues to tumor development. The 5′ HOX genes were aberrantly upregulated during oral carcinogenesis reflecting their posterior prevalence.

A systematic analysis of molecular mechanisms in non-metastatic renal cancer delineates affected regulatory pathways and genes in tumor growth.

In the recent century, Kidney cancer has emerged as one of the critical renal diseases. Therefore, we analyzed gene expression profiles of non-metastatic kidney cancer to find mechanisms associated with the early-stage pathogenesis of the disease. We concentrated on the most dysregulated genes in expression to discover possible unknown proliferative molecular mechanisms and oncogenic pathways promoting kidney renal cancer growth. Survival analysis, expression profiling, and gene set over-representation analysis were conducted on the most upregulated and most down-regulated genes alongside the hub genes. Our results demonstrated that pathways engaged in the metabolism of amino acids and carbohydrates and those involved in peroxisome organization were shown to be important in developing benign tumors. Furthermore, upregulation of genes such as CXCL9 and 10 genes and CXCR4 in chemokine response pathways would bolster differentiation and engagement of immune cells in the tumor microenvironment. C3, one of the essential members of the complement system, with a high degree and betweenness centrality in the PPI network, upregulated significantly not only in our analysis but also in the validation expression profiling results and survival analysis. We also identified genes such as TYROBP, ITGB2, and EGFR to be engaged in both immunological pathways and superoxide pathways. Furthermore, we found that downregulation of Aldolase B engaged in Glycolysis and Gluconeogenesis pathways would help develop benign tumors. Finally, many top hub genes, including TYMS, PTPRC, AURKA, FN1, UBE2C, and CD53 were proposed to be engaged in the progression of non-metastatic renal tumors. This holistic interrogation calls attention to investigate further and experimentally validate the proposed molecular mechanisms.

Attention to time-of-day variability improves the reproducibility of gene expression patterns in multiple sclerosis

SummaryLow reproducibility in gene expression profiles has been observed in transcriptome studies, and this often limits applying findings to clinical practice. Here, we show time-of-day effects on gene expression and analytical schemes to increase the reproducibility in expression patterns. We recruited patients with relapsing-remitting multiple sclerosis (RRMS) and healthy subjects and collected blood from individuals twice a day, day (2 pm) and night (9 pm). RNA sequencing analyses found that gene expression in RRMS in relapse (Relapse) is significantly changed at night compared with either Relapse at day or RRMS in remission (Remission). Gene set overrepresentation analysis demonstrated that gene sets significantly changed in Relapse at night are enriched to immune responses related to MS pathology. In those gene sets, 68 genes are significantly changed expression in Relapse at night compared with Relapse at day and Remission. This supports that times of sample collections should be standardized to obtain reproducible gene expression patterns.

High CD52 mRNA Expression Is An Adverse Prognostic Factor In Fludarabine-Refractory CLL Treated With Alemtuzumab On The Gcllsg CLL2H Trial

Alemtuzumab is a humanized mAb that targets CD52 and has been widely used for the treatment of chronic lymphocytic leukemia (CLL) patients with refractory disease or loss / mutation of TP53. The cell-surface marker CD52 is variably expressed on peripheral blood mononuclear cells and shows relatively high expression levels on the surface of both healthy B lymphocytes and on CLL cells, but the biologic function of CD52 has not been sufficiently clarified yet.To elucidate the prognostic value of CD52 expression, we analyzed a large gene expression dataset. Gene expression profiling was performed on Affymetrix Exon 1.0 ST Arrays. Data sets were generated for 75 fludarabine-refractory CLL patients treated within the multicenter phase II CLL2H trial of the GCLLSG (3x30 mg Alemtuzumab s.c. per week, max. 12 weeks). Normalization of the arrays was conducted by using the RMA algorithm. Statistical analysis was performed with the R software version 2.15.1.Patients were grouped into quartiles (Q1-Q4) according to CD52 expression levels (CD52 low: Q1, n=19; CD52 intermediate: Q2-Q3, n=37; CD52 high: Q4, n=19).Progression free survival (PFS) and overall survival (OS) were markedly different for patients from Q1 and Q4 (median PFS: Q1=413 days vs. Q4=145 days (p=0.0102); median OS: Q1= 879 days vs. Q4=413 days (p=0.0164)). Patients with progressive disease were overrepresented in Q4 (PD 7/18 (38.8%); ORR 5/18 (27.7%)) while rare in Q1 (PD 2/19 (10.5%); ORR 9/19 (47.3%)). Patients with low expression of CD52 had less frequently genetic/biologic and clinical characteristics of aggressive disease (Q1 vs. Q4; TP53 mutation/17p deletion: 47.36% vs. 36.84%; IGHV unmutated: 61.11% vs. 73.68%; leukocyte counts (median): 45.8 G/L vs. 55.4 G/L; thymidine kinase: 22.2 U/L vs. 30.4 U/L; β2-microglobulin: 3.3 mg/l vs. 5.2 mg/l; no palpable lymph nodes: 47.36 % vs. 10.52 %).Mutations of SF3B1 and NOTCH1 were present in 4/17 (23.5%) and in 5/17 (29.4%) cases in Q1. In contrast, cases from Q4 harbored mutations of SF3B1 and NOTCH1 in 2/18 (11.1%) and 1/18 (5.5%).Cases with high CD52 levels showed overexpression (FDR<1e-07;FC>2) of genes predominantly involved in signaling of the B-cell-, toll-like- and interleukin-receptor (BTK, SYK, CD79B; TLR7, TLR10, IL27RA, IL2RG, TRAF3IP3, TRAF5).The corresponding gene set overrepresentation analysis on KEGG pathways was significantly enriched for gene sets related to cell-extracellular matrix interaction, focal adhesion, regulation of actin cytoskeleton and cell adhesion molecules as well as associated signaling pathways like WNT- and MAPK-signaling.In conclusion, high CD52 expression is an adverse prognostic factor in refractory CLL treated with Alemtuzumab. Although suggestive, the level of CD52 expression and subsequent antibody binding capacity appear not to determine the effectivity of treatment. The association with genetic and biological characteristics indicates an independent role in the biology of aggressive CLL and highlights a potential function in cell-microenvironmental interactions for CLL cells. [Display omitted] Disclosures:Stilgenbauer:Genzyme: Honoraria, Research Funding.

Integrative multiomics analysis of human atherosclerosis reveals a serum response factor-driven network associated with intraplaque hemorrhage.

BackgroundWhile single‐omics analyses on human atherosclerotic plaque have been very useful to map stage‐ or disease‐related differences in expression, they only partly capture the array of changes in this tissue and suffer from scale‐intrinsic limitations. In order to better identify processes associated with intraplaque hemorrhage and plaque instability, we therefore combined multiple omics into an integrated model.MethodsIn this study, we compared protein and gene makeup of low‐ versus high‐risk atherosclerotic lesion segments from carotid endarterectomy patients, as judged from the absence or presence of intraplaque hemorrhage, respectively. Transcriptomic, proteomic, and peptidomic data of this plaque cohort were aggregated and analyzed by DIABLO, an integrative multivariate classification and feature selection method.ResultsWe identified a protein‐gene associated multiomics model able to segregate stable, nonhemorrhaged from vulnerable, hemorrhaged lesions at high predictive performance (AUC >0.95). The dominant component of this model correlated with αSMA−PDGFRα+ fibroblast‐like cell content (p = 2.4E‐05) and Arg1+ macrophage content (p = 2.2E‐04) and was driven by serum response factor (SRF), possibly in a megakaryoblastic leukemia‐1/2 (MKL1/2) dependent manner. Gene set overrepresentation analysis on the selected key features of this model pointed to a clear cardiovascular disease signature, with overrepresentation of extracellular matrix synthesis and organization, focal adhesion, and cholesterol metabolism terms, suggestive of the model's relevance for the plaque vulnerability. Finally, we were able to corroborate the predictive power of the selected features in several independent mRNA and proteomic plaque cohorts.ConclusionsIn conclusion, our integrative omics study has identified an intraplaque hemorrhage‐associated cardiovascular signature that provides excellent stratification of low‐ from high‐risk carotid artery plaques in several independent cohorts. Further study revealed suppression of an SRF‐regulated disease network, controlling lesion stability, in vulnerable plaque, which can serve as a scaffold for the design of targeted intervention in plaque destabilization.

Genome-wide association analysis of cognitive function in Danish long-lived individuals

Cognitive function is a substantially heritable trait related to numerous important life outcomes. Several genome-wide association studies of cognitive function have in recent years led to the identification of thousands of significantly associated loci and genes. Individuals included in these studies have rarely been nonagenarians and centenarians, and since cognitive function is an important component of quality of life for this rapidly expanding demographic group, there is a need to explore genetic factors associated with individual differences in cognitive function at advanced ages.In this study, we pursued this by performing a genome-wide association study of cognitive function in 490 long-lived Danes (age range 90.1−100.8 years). While no genome-wide significant SNPs were identified, suggestively significant SNPs (P < 1 × 10−5) were mapped to several interesting genes, including ZWINT, CELF2, and DNAH5, and the glutamate receptor genes GRID2 and GRM7. Additionally, results from a gene set over-representation analysis indicated potential roles of gene sets related to G protein-coupled receptor (GPCR) signaling, interaction between L1 and ankyrins, mitogen-activated protein kinase (MAPK) signaling, RNA degradation, and cell cycle.Larger studies are needed to shed further light on the possible importance of these suggestive genes and pathways in cognitive function in nonagenarians and centenarians.

Abstract PS5-43: Senescence-related methylation changes following therapy as potential biomarker for CDK4/6 inhibitor activity

Abstract IntroductionClassically, cancer therapy approaches lead to tumor cell death or an alternative fate such as senescence. Senescent cells remain viable but without proliferation markers. Senescent cells secrete factors with diverse actions that influence surrounding cells, the extracellular matrix (ECM) and the immune system, a phenomenon termed the senescence-associated secretory phenotype (SASP). Cyclin-dependent kinase 4/6 inhibitors (CDKi) are game-changers in the therapy of metastatic hormone receptor-positive, HER2-negative breast cancer. A potential mechanism of the action of these agents is induction of senescence in breast tumor cells, going beyond cell cycle arrest. However, senescence can be studied mainly using invasive biopsies, and therefore the prevalence and importance of senescence in patients are largely unknown. Moreover, only few circulating biomarkers exist to predict activity or measure CDKi effects. In order to develop a correlative liquid biopsy for CDKi activity, we have analyzed epigenetic (methylation) changes following therapy-induced senescence in vitro. We sought to identify (un)methylated loci that will be affected by different approaches and later can be discovered in circulating cell-free DNA. MethodsWe have treated luminal breast cancer MCF-7 cells with Doxorubicin, CDKi (Palbociclib) or by irradiation. This protocol resulted in up to 80% of cells with senescence-related beta-galactosidase activity. DNA methylation was profiled using Illumina Infinium MethylationEPIC 850K BeadChip. Differentially methylated loci (mDNA) were identified using GenomeStudio and Minfi. Analyses were done using GREAT, CSGene, and Reactome databases. Methylation age was analyzed using the Horvath Methylation Calculator. Statistical significance was defined as p&amp;lt;0.05, q&amp;lt;0.1. ResultsSignificantly differentially methylated sites (in comparison to untreated cells) were revealed as following: 9111 sites in Doxorubicin treated cells, 3828 sites in Palbociclib treated cells, and 694 sites in irradiated cells. These loci comprise 1%, 0.45%, and 0.08% of analyzed methylation sites, respectively. 324 loci were similarly changed following the three treatment options ('common sites'). We found that the 'geographic' distribution of intragenic and intergenic methylation sites (5' UTR, gene body etc.) in all treated cells was similar. Gene set over-representation analysis revealed that sites associated with genes of the 'collagen metabolic process' set were significantly altered in drug-treated cells. Pathway investigation of the 324 'common sites' revealed that ECM-related 'focal adhesion assembly' is the most significant pathway involved. Specific analysis of senescence-related gene sets showed that 15.1% (76/503) of senescence genes changed after Doxorubicin treatment, 6.2% (31/503) after Palbociclib and 1% (5/503) after irradiation. Pathway analysis of these genes showed that drugs affected the 'oxidative stress-induced senescence' pathway, while irradiated cells had SASP-related genes affected.Despite the above mentioned methylation changes, age calculation based on methylation clock showed that all samples had similar age (6), regardless of manipulation. ConclusionSingle treatment of MCF-7 cells with known senescence inducers results in changes in methylation patterns. A significant number of common loci changed following all types of treatments, suggesting them as potential surrogate loci of senescence. Also, we have reproduced the well-known interplay between collagen and senescence/SASP.This preliminary data sheds light on epigenetic changes following treatment-induced senescence. Further studies are needed to validate whether these methylation changes can be found in vivo in tumors and in patients’ cell-free DNA following therapy with CDKi. Citation Format: Albert Grinshpun, Anatoli Kustanovich, Joshua Moss, Yael Gabai, Yuval Dor, Ittai Ben-Porath. Senescence-related methylation changes following therapy as potential biomarker for CDK4/6 inhibitor activity [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS5-43.

An Epigenome-Wide Analysis of Ambient Pyrethroid Pesticide Exposures in California's Central Valley

Background/Aim Pyrethroid pesticide use is increasing worldwide, although the full extent of associated health effects is unknown. An epigenome-wide analysis study (EWAS) with exploratory pathway analysis may help identify potential pyrethroid-related health effects.Methods We performed an EWAS of chronic ambient pyrethroid exposure using control participants’ blood in the Parkinson’s Environment and Genes Study in California (N=237). We estimated associations of exposure to ambient pyrethroid pesticide applications in the 5 years prior to enrollment with differential methylation at enrollment, using beta regression and an FDR q &lt;0.05 for significance. We normalized methylation values for typeI/II probe bias using BMIQ, evaluated batch effects with SVA, and adjusted for cell count. We also performed gene set overrepresentation analysis on the genes annotated to CpG sites that were associated with pyrethroids at a raw p-value cutoff of 0.05. For gene-set overrepresentation analyses, we controlled for background counts of CpG sites on the Illumina450K chip, and identified Gene Ontology (GO) biological process terms with missMethyl, and OMIM and Glad4U disease-associated gene sets. We used an FDR q&lt; 0.05 to evaluate statistical significance of gene sets. Results 5 CpG sites were differentially methylated in relation to pyrethroid exposures. Two of these sites annotated to genes involved in calcium ion binding, a known primary target of pyrethroid pesticides. We also identified 40 GO terms, 14 of which were neurological/developmental in nature. For disease sets, we identified signals for Alzheimer’s disease, leukemia and several other cancers, diabetes, birth defects, and other diseases. Conclusions Chronic ambient pyrethroid exposure is associated with differential methylation at CpG sites that annotate to a wide variety of disease states and biological mechanisms. While several of the identified diseases and gene processes are consistent with prior research, this EWAS also implicates several previously unidentified diseases for future investigation in relation to pyrethroid exposure.

An epigenome-wide association study of ambient pyrethroid pesticide exposures in California's central valley.

Pyrethroid pesticide use is increasing worldwide, although the full extent of associated health effects is unknown. An epigenome-wide association study (EWAS) with exploratory pathway analysis may help identify potential pyrethroid-related health effects. We performed an exploratory EWAS of chronic ambient pyrethroid exposure using control participants' blood in the Parkinson's Environment and Genes Study in the Central Valley of California (N=237). We estimated associations of living and working near agricultural pyrethroid pesticide applications in the past 5 years (binary) with site-specific differential methylation, and used a false discovery rate (FDR) cut off of 0.05 for significance. We controlled for age, sex, education, cell count, and an ancestral marker for Hispanic ethnicity. We normalized methylation values for Type I/II probe bias using Beta-Mixture Quantile (BMIQ) normalization, filtered out cross-reactive probes, and evaluated for remaining bias with Surrogate Variable Analysis (SVA). We also evaluated the effects of controlling for cell count and normalizing for Type I/II probe bias by comparing changes in effect estimates and p-values for the top hits across BMIQ and GenomeStudio normalization methods, and controlling for cell count. To facilitate broader interpretation, we annotated genes to the CpG sites and performed gene set overrepresentation analysis, using genes annotated to CpG sites that were associated with pyrethroids at a raw p<0.05, and controlling for background representation of CpG sites on the chip. We did this for both a biological process context (Gene Ontology terms) using missMethyl, and a disease set context using WebGestalt. For these gene set overrepresentation analyses we also used an FDR cut off of 0.05 for significance of gene sets. After controlling for cell count and applying BMIQ normalization, 4 CpG sites were differentially methylated in relation to pyrethroid exposures. When using GenomeStudio's Illumina normalization, 415 CpG sites were differentially methylated, including all four identified with the BMIQ method. In the gene set overrepresentation analyses, we identified 6 GO terms using BMIQ normalization, and 76 using Illumina normalization, including the 6 identified by BMIQ. For disease sets, we identified signals for Alzheimer's disease, leukemia and several other cancers, diabetes, birth defects, and other diseases, for both normalization methods. We identified minimal changes in effect estimates after controlling for cell count, and controlling for cell count generally weakened p-values. BMIQ normalization, however, resulted in different beta coefficients and weakened p-values. Chronic ambient pyrethroid exposure is associated with differential methylation at CpG sites that annotate to a wide variety of disease states and biological mechanisms that align with prior research. However, this EWAS also implicates several novel diseases for future investigation, and highlights the relative importance of different background normalization methods in identifying associations.

GSOAP: a tool for visualization of gene set over-representation analysis.

MotivationGene sets over-representation analysis (GSOA) is a common technique of enrichment analysis that measures the overlap between a gene set and selected instances (e.g. pathways). Despite its popularity, there is currently no established standard for visualization of GSOA results.ResultsHere, we propose a visual exploration of the GSOA results by showing the relationships among the enriched instances, while highlighting important instance attributes, such as significance, closeness (centrality) and clustering.Availability and implementationGSOAP is implemented as an R package and is available at https://github.com/tomastokar/gsoap.

Schwann cell reprogramming and lung cancer progression: a meta-analysis of transcriptome data.

Schwann cells were identified in the tumor surrounding area prior to initiate the invasion process underlying connective tissue. These cells promote cancer invasion through direct contact, while paracrine signaling and matrix remodeling are not sufficient to proceed. Considering the intertwined structure of signaling, regulatory, and metabolic processes within a cell, we employed a genome-scale biomolecular network. Accordingly, a meta-analysis of Schwann cells associated transcriptomic datasets was performed, and the core information on differentially expressed genes (DEGs) was obtained by statistical analyses. Gene set over-representation analyses was performed on core DEGs to identify significantly functional and pathway enrichment analysis between Schwann cells and, lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC). DEGs were further integrated with genome-scale human biomolecular networks. miRNAs were proposed by the reconstruction of a transcriptional and post-transcriptional regulatory network. Moreover, microarray-based transcriptome profiling was performed, and the prognostic power of selected dedifferentiated Schwann cell biomolecules was predicted. We observed that pathways associated with Schwann cells dedifferentiation was overexpressed in lung cancer samples. However, genes associated with Schwann cells migration inhibition system were downregulated. Besides, miRNA targeting those pathways were also deregulated. In this study, we report valuable data for further experimental and clinical analysis, because the proposed biomolecules have significant potential as systems biomarkers for screening or for therapeutic purposes in perineural invasion of lung cancer.

Copy number variation-based gene set analysis reveals cytokine signalling pathways associated with psychiatric comorbidity in patients with inflammatory bowel disease

BackgroundRecent studies discovered many genetic variants associated with both psychiatric and inflammatory disorders, but the role of genetic factors in the development of psychiatric comorbidity (PC) in inflammatory bowel disease (IBD) is underexplored. Particularly, it has been shown that some of the genetic variants have been linked to the concentrations of circulating cytokines and symptoms of the inflammatory cytokine-associated depression. We analysed genomic features of individuals with IBD by comparing IBD patients with PC with those who have IBD but without PC. We hypothesized that cytokine related signalling pathways may be significantly associated with the psychiatric comorbidity in patients with IBD. MethodsIndividuals enrolled in the Manitoba IBD Cohort Study were separated to two groups accordingly to the presence of PC. A sample set comprising 97 IBD individuals with PC (IBD + PC) and 146 IBD individuals without PC (IBD) was first used to identify copy number variations (CNVs) from genome-wide genetic data using three different detection algorithms. IBD + PC and IBD groups were compared by the number of CNVs overlapping each gene; deletions and duplications were analysed separately. Gene set overrepresentation analysis was then conducted using CNV-overlapped genes and the candidate gene sets of neurological and immunological relevance. ResultsMedium-sized CNV (size between 100 and 500 kilobase pairs)-burden is significantly higher in IBD + PC than IBD groups. Gene-based CNV association analysis did not show significant differences between the two IBD groups. Gene set overrepresentation analysis demonstrated the significant enrichment of gene sets related to cytokine signalling pathways by the genes overlapped by deletions in the IBD individuals with PC. ConclusionOur results confirm the role of cytokine signalling pathways in the development of PC in IBD. Additionally, our results warrant further study with a larger sample size focusing on cytokine SNPs to further understand the relationship between inflammatory and psychiatric disorders.

A MIR4646 associated methylation locus is hypomethylated in adolescent depression

BackgroundStudies of epigenetics and transcriptional activity in adolescents may provide knowledge about possible preventive strategies of depression. MethodsWe present a methylome-wide association study (MWAS) and cohort validation analysis of depression in adolescents, in two separate cohorts: discovery (n=93) and validation data set 1 (n=78). The genome-wide methylation pattern was measured from whole blood using the Illumina 450K array. A second validation cohort, validation data set 2, consists of post-mortem brain biopsies from depressed adults (n=58). We performed a MWAS by robust multiple linear regressions of methylation to a modified risk-score assessment of depression. Methylation levels of candidate CpG sites were correlated with expression levels of the associated gene in an independent cohort of 11 healthy volunteers. ResultsThe methylation state of two CpG sites reliably predicted ratings of depression in adolescents (cg13227623 and cg04102384) (p<10E-06). Cohort validation analysis confirmed cg04102384 – located in the promoter region of microRNA 4646 (MIR4646) – to be hypomethylated in both validation data set 1 and validation data set 2 (p<0.05). Cg04102384 was inversely correlated to expression levels of MIR4646-3p in healthy controls (p<0.05). LimitationsMIR4646 was not differentially expressed in a subset of samples with adolescent depression measured by qRT-PCR measurements. ConclusionWe identify a specific MIR4646 associated epigenetic risk site to be associated with depression in adolescents. Cg04102384 putatively regulates gene expression of MIR4646-3p. Target gene prediction and gene set overrepresentation analysis revealed involvement of this miRNA in fatty acid elongation, a process related to omega-3 fatty acids, previously associated with depression.