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.
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.
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.
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.
Objective
Heightened inflammation, dysregulated immunity, and thrombotic events are characteristic of hospitalized COVID‐19 patients. Given that platelets are key regulators of thrombosis, ...inflammation, and immunity they represent prime candidates as mediators of COVID‐19‐associated pathogenesis. The objective of this study was to understand the contribution of severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) to the platelet phenotype via phenotypic (activation, aggregation) and transcriptomic characterization.
Approach and Results
In a cohort of 3915 hospitalized COVID‐19 patients, we analyzed blood platelet indices collected at hospital admission. Following adjustment for demographics, clinical risk factors, medication, and biomarkers of inflammation and thrombosis, we find platelet count, size, and immaturity are associated with increased critical illness and all‐cause mortality. Bone marrow, lung tissue, and blood from COVID‐19 patients revealed the presence of SARS‐CoV‐2 virions in megakaryocytes and platelets. Characterization of COVID‐19 platelets found them to be hyperreactive (increased aggregation, and expression of P‐selectin and CD40) and to have a distinct transcriptomic profile characteristic of prothrombotic large and immature platelets. In vitro mechanistic studies highlight that the interaction of SARS‐CoV‐2 with megakaryocytes alters the platelet transcriptome, and its effects are distinct from the coronavirus responsible for the common cold (CoV‐OC43).
Conclusions
Platelet count, size, and maturity associate with increased critical illness and all‐cause mortality among hospitalized COVID‐19 patients. Profiling tissues and blood from COVID‐19 patients revealed that SARS‐CoV‐2 virions enter megakaryocytes and platelets and associate with alterations to the platelet transcriptome and activation profile.
Estrogen receptor α (ER) ligand-binding domain (LBD) mutations are found in a substantial number of endocrine treatment-resistant metastatic ER-positive (ER+) breast cancers. We investigated the ...chromatin recruitment, transcriptional network, and genetic vulnerabilities in breast cancer models harboring the clinically relevant ER mutations. These mutants exhibit both ligand-independent functions that mimic estradiol-bound wild-type ER as well as allele-specific neomorphic properties that promote a pro-metastatic phenotype. Analysis of the genome-wide ER binding sites identified mutant ER unique recruitment mediating the allele-specific transcriptional program. Genetic screens identified genes that are essential for the ligand-independent growth driven by the mutants. These studies provide insights into the mechanism of endocrine therapy resistance engendered by ER mutations and potential therapeutic targets.
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•The mutant ER cistromes are different from the E2-stimulated WT ER cistrome•The ER mutant-selective cistromes drive endocrine resistance and metastases•The ER Y537S and D538G mutants have distinct cistromes and transcriptomes•THZ1 blocks mutant ER phosphorylation at S118 and inhibits mutant ER cell growth
Jeselsohn et al. show that estrogen receptor α (ER) mutations found in endocrine treatment-resistant metastatic breast cancers confer not only ligand-independent ER functions, but also allele-specific neomorphic properties. Importantly, the authors identify potential approaches for treating these breast cancers.
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.
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.
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.
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.
Background
Psoriasis is an inflammatory skin disease associated with increased cardiovascular (CV) risk, whose pathogenesis is not fully known.
Objective
We identified a transcriptomic signature in ...psoriasis and investigated its association with prevalent and future risk of a CV event to understand the connection between psoriasis and CV disease (CVD).
Methods
Psoriasis patients (n = 37) with a history of moderate–severe skin disease without CVD and 11 matched controls underwent whole blood RNA sequencing. This transcriptomic signature in psoriasis versus controls was evaluated in two CVD cohorts: Women referred for cardiac catheterization with (n = 76) versus without (n = 97) myocardial infarction (MI), and patients with peripheral artery disease (n = 106) followed over 2.5 years for major adverse CV or limb events (MACLE). The association between genes differentially expressed in psoriasis and prevalent and incident CV events was assed.
Results
In psoriasis, median age was 44 (IQR; 34–51) years, 49% male and ACC/AHA ASCVD Risk Score of 1.0% (0.6–3.4) with no significant difference versus controls. The median psoriasis area and severity index score (PASI) was 4.0 (IQR 2.9–8.2) with 36% on biologic therapy. Overall, 247 whole blood genes were upregulated and 228 downregulated in psoriasis versus controls (p < 0.05), and 1302 genes positively and 1244 genes negatively correlated with PASI (p < 0.05). Seventy‐three genes overlapped between psoriasis prevalence and PASI with key regulators identified as IL‐6, IL‐1β and interferon gamma. In the CVD cohorts, 50 of 73 genes (68%) identified in psoriasis associated with prevalent MI, and 29 (40%) with incident MACLE. Key regulator transcripts identified in psoriasis and CVD cohorts included SOCS3, BCL3, OSM, PIM2, PIM3 and STAT5A.
Conclusions
A whole blood transcriptomic signature of psoriasis diagnosis and severity associated with prevalent MI and incident MACLE. These data have implications for better understanding the link between psoriasis, systemic inflammation and CVD.
Objective
Platelets are mediators of inflammation with immune effector cell properties and have been implicated in the pathogenesis of systemic lupus erythematosus (SLE). This study investigated the ...role of platelet‐associated lectin, galactoside‐binding, soluble 3 binding protein (LGALS3BP) as a mediator of inflammation in SLE and as a potential biomarker associated with clinical phenotypes.
Methods
We performed RNA sequencing on platelets from patients with SLE (n = 54) and on platelets from age‐, sex‐, and race/ethnicity‐matched healthy controls (n = 18) and measured LGALS3BP levels in platelet releasate and in circulating serum. We investigated the association between LGALS3BP levels and the prevalence, disease severity, and clinical phenotypes of SLE and studied platelet‐mediated effects on myeloid inflammation.
Results
Platelets from patients with SLE exhibited increased expression of LGALS3BP (fold change 4.0, adjusted P = 6.02 × 10−11). Platelet‐released LGALS3BP levels were highly correlated with circulating LGALS3BP (R = 0.69, P < 0.0001), and circulating LGALS3BP levels were correlated with the severity of disease according to the SLE Disease Activity Index (r = 0.32, P = 0.0006). Specifically, circulating LGALS3BP levels were higher in SLE patients with lupus nephritis than in patients with inactive disease (4.0 μg/ml versus 2.3 μg/ml; P < 0.001). Interferon‐α induced LGALS3BP transcription and translation in a megakaryoblastic cell line (MEG‐01) in a dose‐dependent manner. Recombinant LGALS3BP and platelet releasates from SLE patients enhanced proinflammatory cytokine production by macrophages.
Conclusions
Our results support that platelets act as potent effector cells that contribute to the pathogenesis of SLE by secreting proinflammatory LGALS3BP, which also represents a novel biomarker of SLE clinical activity.
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 (Lpa), 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.
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