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Background RNA sequencing has become a ubiquitous technology used throughout life sciences as an effective method of measuring RNA abundance quantitatively in tissues and cells. The increase in use of RNA-seq technology has led to the continuous development of new tools for every step of analysis from alignment to downstream pathway analysis. However, effectively using these analysis tools in a scalable and reproducible way can be challenging, especially for non-experts. Results Using the workflow management system Snakemake we have developed a user friendly, fast, efficient, and comprehensive pipeline for RNA-seq analysis. VIPER (Visualization Pipeline for RNA-seq analysis) is an analysis workflow that combines some of the most popular tools to take RNA-seq analysis from raw sequencing data, through alignment and quality control, into downstream differential expression and pathway analysis. VIPER has been created in a modular fashion to allow for the rapid incorporation of new tools to expand the capabilities. This capacity has already been exploited to include very recently developed tools that explore immune infiltrate and T-cell CDR (Complementarity-Determining Regions) reconstruction abilities. The pipeline has been conveniently packaged such that minimal computational skills are required to download and install the dozens of software packages that VIPER uses. Conclusions VIPER is a comprehensive solution that performs most standard RNA-seq analyses quickly and effectively with a built-in capacity for customization and expansion.

CTCF and cohesin play a key role in organizing chromatin into topologically associating domain (TAD) structures. Disruption of a single CTCF binding site is sufficient to change chromosomal interactions leading to alterations in chromatin modifications and gene regulation. However, the extent to which alterations in chromatin modifications can disrupt 3D chromosome organization leading to transcriptional changes is unknown. In multiple myeloma, a 4;14 translocation induces overexpression of the histone methyltransferase, NSD2, resulting in expansion of H3K36me2 and shrinkage of antagonistic H3K27me3 domains. Using isogenic cell lines producing high and low levels of NSD2, here we find oncogene activation is linked to alterations in H3K27ac and CTCF within H3K36me2 enriched chromatin. A logistic regression model reveals that differentially expressed genes are significantly enriched within the same insulated domain as altered H3K27ac and CTCF peaks. These results identify a bidirectional relationship between 2D chromatin and 3D genome organization in gene regulation. In multiple myeloma, a 4;14 translocation induces overexpression of histone methyltransferase NSD2, resulting in expansion of H3K36me2 and shrinkage of H3K27me3 domains. Here the authors find that CTCF, H3K27ac and gene expression changes cluster within a subset of insulated domains implicating 3D chromosome organization as a key factor in the NSD2-mediated phenotype.

Background The clinical heterogeneity of SLE with its complex pathogenesis remains challenging as we strive to provide optimal management. The contribution of platelets to endovascular homeostasis, inflammation and immune regulation highlights their potential importance in SLE. Prior work from our group showed that the Fcγ receptor type IIa (FcγRIIa)–R/H131 biallelic polymorphism is associated with increased platelet activity and cardiovascular risk in SLE. The study was initiated to investigate the platelet transcriptome in patients with SLE and evaluate its association across FcγRIIa genotypes and distinct clinical features. Methods Fifty-one patients fulfilling established criteria for SLE (mean age = 41.1 ± 12.3, 100% female, 45% Hispanic, 24% black, 22% Asian, 51% white, mean SLEDAI = 4.4 ± 4.2 at baseline) were enrolled and compared with 18 demographically matched control samples. The FCGR2a receptor was genotyped for each sample, and RNA-seq was performed on isolated, leukocyte-depleted platelets. Transcriptomic data were used to create a modular landscape to explore the differences between SLE patients and controls and various clinical parameters in the context of FCGR2a genotypes. Results There were 2290 differentially expressed genes enriched for pathways involved in interferon signaling, immune activation, and coagulation when comparing SLE samples vs controls. When analyzing patients with proteinuria, modules associated with oxidative phosphorylation and platelet activity were unexpectedly decreased. Furthermore, genes that were increased in SLE and in patients with proteinuria were enriched for immune effector processes, while genes increased in SLE but decreased in proteinuria were enriched for coagulation and cell adhesion. A low-binding FCG2Ra allele (R131) was associated with decreases in FCR activation, which further correlated with increases in platelet and immune activation pathways. Finally, we were able to create a transcriptomic signature of clinically active disease that performed significantly well in discerning SLE patients with active clinical disease form those with inactive clinical disease. Conclusions In aggregate, these data demonstrate the platelet transcriptome provides insight into lupus pathogenesis and disease activity, and shows potential use as means of assessing this complex disease using a liquid biopsy.

Platelets are key mediators of atherothrombosis, yet, limited tools exist to identify individuals with a hyperreactive platelet phenotype. In this study, we investigate the association of platelet hyperreactivity and cardiovascular events, and introduce a tool, the Platelet Reactivity ExpreSsion Score (PRESS), which integrates platelet aggregation responses and RNA sequencing. Among patients with peripheral artery disease (PAD), those with a hyperreactive platelet response (>60% aggregation) to 0.4 µM epinephrine had a higher incidence of the 30 day primary cardiovascular endpoint (37.2% vs. 15.3% in those without hyperreactivity, adjusted HR 2.76, 95% CI 1.5–5.1, p  = 0.002). PRESS performs well in identifying a hyperreactive phenotype in patients with PAD (AUC [cross-validation] 0.81, 95% CI 0.68 –0.94, n  = 84) and in an independent cohort of healthy participants (AUC [validation] 0.77, 95% CI 0.75 –0.79, n  = 35). Following multivariable adjustment, PAD individuals with a PRESS score above the median are at higher risk for a future cardiovascular event (adjusted HR 1.90, CI 1.07–3.36; p  = 0.027, n  = 129, NCT02106429). This study derives and validates the ability of PRESS to discriminate platelet hyperreactivity and identify those at increased cardiovascular risk. Future studies in a larger independent cohort are warranted for further validation. The development of a platelet reactivity expression score opens the possibility for a personalized approach to antithrombotic therapy for cardiovascular risk reduction. Platelet hyperreactivity is associated with cardiovascular events in patients with PAD. Here the authors derive and validate a circulating platelet genetic signature to discriminate platelet hyperreactivity and cardiovascular risk.

Myocardial injury after non-cardiac surgery (MINS) is common. We investigated the incidence and outcomes of MINS, and mechanistic underpinnings using pre-operative whole blood gene expression profiling in a prospective cohort study of individuals undergoing lower extremity revascularization (LER) for peripheral artery disease (PAD). Major adverse cardiovascular and limb events (MACLE) were defined as a composite of death, myocardial infarction, stroke, major lower extremity amputation or reoperation. Among 226 participants undergoing LER, MINS occurred in 53 (23.5%). Patients with MINS had a greater incidence of major adverse cardiovascular events (49.1% vs. 22.0%, adjusted HR 1.87, 95% CI 1.07–3.26) and MACLE (67.9% vs. 44.5%; adjusted HR 1.66, 95% CI 1.08–2.55) at median 20-month follow-up. Pre-operative whole blood transcriptome profiling of a nested matched MINS case–control cohort (n = 41) identified upregulation of pathways related to platelet alpha granules and coagulation in patients who subsequently developed MINS. Thrombospondin 1 ( THBS1 ) mRNA expression was 60% higher at baseline in patients who later developed MINS, and was independently associated with long-term cardiovascular events in the Duke Catheterization Genetics biorepository cohort. In conclusion, pre-operative THBS1 mRNA expression is higher in patients who subsequently develop MINS and is associated with incident cardiovascular events. Pathways related to platelet activity and coagulation associated with MINS provide novel insights into mechanisms of myocardial injury.

The estrogen receptor (ER) drives the growth of most luminal breast cancers and is the primary target of endocrine therapy. Although ER blockade with drugs such as tamoxifen is very effective, a major clinical limitation is the development of endocrine resistance especially in the setting of metastatic disease. Preclinical and clinical observations suggest that even following the development of endocrine resistance, ER signaling continues to exert a pivotal role in tumor progression in the majority of cases. Through the analysis of the ER cistrome in tamoxifen-resistant breast cancer cells, we have uncovered a role for an RUNX2–ER complex that stimulates the transcription of a set of genes, including most notably the stem cell factor SOX9, that promote proliferation and a metastatic phenotype. We show that up-regulation of SOX9 is sufficient to cause relative endocrine resistance. The gain of SOX9 as an ER-regulated gene associated with tamoxifen resistance was validated in a unique set of clinical samples supporting the need for the development of improved ER antagonists.

Biomarkers may improve prediction of cardiovascular events for patients with stable coronary artery disease (CAD), but their importance in addition to clinical tests of inducible ischemia and CAD severity is unknown. To evaluate the prognostic value of multiple biomarkers in stable outpatients with obstructive CAD and moderate or severe inducible ischemia. The ISCHEMIA and ISCHEMIA CKD trials randomized 5,956 participants with CAD to invasive or conservative management from July 2012 to January 2018; 1,064 participated in the biorepository. Primary outcome was cardiovascular death, myocardial infarction (MI), or hospitalization for unstable angina, heart failure, or resuscitated cardiac arrest. Secondary outcome was cardiovascular death or MI. Improvements in prediction were assessed by cause-specific hazard ratios (HR) and area under the receiver operating characteristics curve (AUC) for an interquartile increase in each biomarker, controlling for other biomarkers, in a base clinical model of risk factors, left ventricular ejection fraction (LVEF) and ischemia severity. Secondary analyses were performed among patients in whom core-lab confirmed severity of CAD was ascertained by computed cardiac tomographic angiography (CCTA). Baseline levels of interleukin-6 (IL-6), high sensitivity troponin T (hsTnT), growth differentiation factor 15 (GDF-15), N-terminal pro-B-type natriuretic peptide (NT-proBNP), lipoprotein a (Lp[a]), high sensitivity C-reactive protein (hsCRP), Cystatin C, soluble CD 40 ligand (sCD40L), myeloperoxidase (MPO), and matrix metalloproteinase 3 (MMP3). Among 757 biorepository participants, median (IQR) follow-up was 3 (2-5) years, age was 67 (61-72) years, and 144 (19%) were female; 508 had severity of CAD by CCTA available. In an adjusted multimarker model with hsTnT, GDF-15, NT-proBNP and sCD40L, the adjusted HR for the primary outcome per interquartile increase in each biomarker was 1.58 (95% CI 1.22, 2.205), 1.60 (95% CI 1.16, 2.20), 1.61 (95% 1.22, 2.14), and 1.46 (95% 1.12, 1.90), respectively. The adjusted multimarker model also improved prediction compared with the clinical model, increasing the AUC from 0.710 to 0.792 (P < .01) and 0.714 to 0.783 (P < .01) for the primary and secondary outcomes, respectively. Similar findings were observed after adjusting for core-lab confirmed atherosclerosis severity. Among ISCHEMIA biorepository participants, biomarkers of myocyte injury/distension, inflammation, and platelet activity improved cardiovascular event prediction in addition to risk factors, LVEF, and assessments of ischemia and atherosclerosis severity. These biomarkers may improve risk stratification for patients with stable CAD.

Activation of regulatory elements is thought to be inversely correlated with DNA methylation levels. However, it is difficult to determine whether DNA methylation is compatible with chromatin accessibility or transcription factor (TF) binding if assays are performed separately. We developed a fast, low-input, low sequencing depth method, EpiMethylTag, that combines ATAC-seq or ChIP-seq (M-ATAC or M-ChIP) with bisulfite conversion, to simultaneously examine accessibility/TF binding and methylation on the same DNA. Here we demonstrate that EpiMethylTag can be used to study the functional interplay between chromatin accessibility and TF binding (CTCF and KLF4) at methylated sites.

BackgroundSLE is an inflammatory condition associated with hyperactivation of the immune system, with mounting evidence that imbalances in the gut microbiota communities are common. These imbalances can range from subtle patterns of dysbiosis to blooms in abundance of individual species that are concordant with clinical disease flares. Based on preliminary longitudinal surveys, almost half of Lupus nephritis (LN) flares were concurrent with transient expansions of a pathobiont, Ruminococcus (blautia) gnavus. As the transcriptomic patterns in the cells in our bloodstream can reflect disease activity, we sought to investigate gene expression patterns in groups of lupus patients, with comparisons to healthy controls (HC).MethodsFrom a well-characterized cohort, exploratory studies were performed on a selected group of 15 active female SLE patients, based in part on SLEDAI scores > 4. Patients were grouped as without a history of renal involvement (i.e., non-renal) (N=7) or with LN in flare with Urine Protein creatinine ratio >0.5. Based on 16S rRNA fecal microbiota analyses, LN were subsetted as without bloom of individual species (N=4), or with a bloom ( > 20-fold increased from HC, 3-9% abundance) of R. gnavus (N=4). In comparisons with 8 female HC, bulk RNA-seq was completed and after standard QC and filtering, 24,319 genes passed a minimum threshold of 4 reads in >50% samples and were used in downstream analysis.ResultsUnsupervised clustering based on gene expression demonstrated significant separation between SLE samples and HC (figure 1). While there was no clear distinction between non-renal and renal (i.e., LN) groups, there were striking differences in the group of active LN without detectable gut microbiome blooms vs. active LN with R. gnavus blooms (figure 2), with 173 upregulated and 13 downregulated genes based on differential expression analysis (padj < 0.05, log2fc > 1). Gene set enrichment analysis (GSEA) identified several significantly altered pathways in the LN flare with blooms compared to no blooms, in highlighting multiple platelet activation pathways in the LN with blooms. In contrast, the LN flares without blooms had significantly higher interferon alpha and interferon gamma signatures.ConclusionOur findings document two major types of flares of LN, with one being mediated by higher PBMC IFN- type I and -gamma transcript levels, and a second without this signature that instead is dominated by several pathways for platelet activation that can be responsible for systemic thromboinflammation. This second distinct pathways of immune activation in PBMC of LN in flare was concurrent with gut blooms of R. gnavus, a pathobiont that induces increased gut permeability, systemic inflammation, bacterial translocation and autoantibody production, which has a well-established association with active Lupus Nephritis. These findings may indicate that in a major subset of patients, LN flares may arise from severe perturbations of intestinal communities associated with a leaky gut, Together, this highlights R. gnavus as a potential causative agent for Lupus flares, in which we hypothesize that gut leak of innate immune stimuli, such as TLR2 and TLR4, which activate platelets in immune hyperactivation pathways that underlie lupus autoimmune pathogenesis.Abstract 1103 Figure 1Principal Component Analysis show differences in gene expression of 15 female SLE patients and 8 healthy female controls.Abstract 1103 Figure 2Differential gene representation in the two LN flare groups compared with controls. Here, we have identified the gene transcripts with altered response to LN in the R. gnavus bloom (red circle).

ESR1 mutations were recently found to be an important mechanism of endocrine resistance in ER-positive (ER + ) metastatic breast cancer. To determine the clinicopathological features driving the emergence of the ESR1 mutations we studied plasma cfDNA and detailed clinical data collected from patients with metastatic breast cancer. Droplet Digital PCR was performed for the detection of the most common ESR1 mutations and PIK3CA mutations. Among the patients with ER + /HER2- disease, ESR1 mutations were detected in 30% of the patients. There were no associations between the pathological features of the primary disease or time to distant recurrence and the emergence of ESR1 mutations in metastatic disease. The prevalence of the ESR1 mutations was significantly associated with prior treatment with an aromatase inhibitor in the adjuvant or metastatic setting. The prevalence of the ESR1 mutations was also positively associated with prior fulvestrant treatment. Conversely, the prevalence of ESR1 mutations was lower after treatment with a CDK4/6 inhibitor. There were no significant associations between specific systemic treatments and the prevalence of PIK3CA mutations. These results support the evolution of the ESR1 mutations under the selective pressure of treatment with aromatase inhibitors in the adjuvant and metastatic settings and have important implications in the optimization of adjuvant and metastatic treatment in ER + breast cancer.