Allelic expression mapping across cellular lineages to establish impact of non-coding SNPs

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Canagliflozin and Cardiovascular and Renal Outcomes in Type 2 Diabetes Mellitus and Chronic Kidney Disease in Primary and Secondary Cardiovascular Prevention Groups

Background: Canagliflozin reduces the risk of kidney failure in patients with type 2 diabetes mellitus and chronic kidney disease, but effects on specific cardiovascular outcomes are uncertain, as are effects in people without previous cardiovascular disease (primary prevention). Methods: In CREDENCE (Canagliflozin and Renal Events in Diabetes With Established Nephropathy Clinical Evaluation), 4401 participants with type 2 diabetes mellitus and chronic kidney disease were randomly assigned to canagliflozin or placebo on a background of optimized standard of care. Results: Primary prevention participants (n=2181, 49.6%) were younger (61 versus 65 years), were more often female (37% versus 31%), and had shorter duration of diabetes mellitus (15 years versus 16 years) compared with secondary prevention participants (n=2220, 50.4%). Canagliflozin reduced the risk of major cardiovascular events overall (hazard ratio [HR], 0.80 [95% CI, 0.67-0.95]; P=0.01), with consistent reductions in both the primary (HR, 0.68 [95% CI, 0.49-0.94]) and secondary (HR, 0.85 [95% CI, 0.69-1.06]) prevention groups (P for interaction=0.25). Effects were also similar for the components of the composite including cardiovascular death (HR, 0.78 [95% CI, 0.61-1.00]), nonfatal myocardial infarction (HR, 0.81 [95% CI, 0.59-1.10]), and nonfatal stroke (HR, 0.80 [95% CI, 0.56-1.15]). The risk of the primary composite renal outcome and the composite of cardiovascular death or hospitalization for heart failure were also consistently reduced in both the primary and secondary prevention groups (P for interaction >0.5 for each outcome). Conclusions: Canagliflozin significantly reduced major cardiovascular events and kidney failure in patients with type 2 diabetes mellitus and chronic kidney disease, including in participants who did not have previous cardiovascular disease

Canagliflozin and renal outcomes in type 2 diabetes and nephropathy

BACKGROUND Type 2 diabetes mellitus is the leading cause of kidney failure worldwide, but few effective long-term treatments are available. In cardiovascular trials of inhibitors of sodium–glucose cotransporter 2 (SGLT2), exploratory results have suggested that such drugs may improve renal outcomes in patients with type 2 diabetes. METHODS In this double-blind, randomized trial, we assigned patients with type 2 diabetes and albuminuric chronic kidney disease to receive canagliflozin, an oral SGLT2 inhibitor, at a dose of 100 mg daily or placebo. All the patients had an estimated glomerular filtration rate (GFR) of 30 to <90 ml per minute per 1.73 m2 of body-surface area and albuminuria (ratio of albumin [mg] to creatinine [g], >300 to 5000) and were treated with renin–angiotensin system blockade. The primary outcome was a composite of end-stage kidney disease (dialysis, transplantation, or a sustained estimated GFR of <15 ml per minute per 1.73 m2), a doubling of the serum creatinine level, or death from renal or cardiovascular causes. Prespecified secondary outcomes were tested hierarchically. RESULTS The trial was stopped early after a planned interim analysis on the recommendation of the data and safety monitoring committee. At that time, 4401 patients had undergone randomization, with a median follow-up of 2.62 years. The relative risk of the primary outcome was 30% lower in the canagliflozin group than in the placebo group, with event rates of 43.2 and 61.2 per 1000 patient-years, respectively (hazard ratio, 0.70; 95% confidence interval [CI], 0.59 to 0.82; P=0.00001). The relative risk of the renal-specific composite of end-stage kidney disease, a doubling of the creatinine level, or death from renal causes was lower by 34% (hazard ratio, 0.66; 95% CI, 0.53 to 0.81; P<0.001), and the relative risk of end-stage kidney disease was lower by 32% (hazard ratio, 0.68; 95% CI, 0.54 to 0.86; P=0.002). The canagliflozin group also had a lower risk of cardiovascular death, myocardial infarction, or stroke (hazard ratio, 0.80; 95% CI, 0.67 to 0.95; P=0.01) and hospitalization for heart failure (hazard ratio, 0.61; 95% CI, 0.47 to 0.80; P<0.001). There were no significant differences in rates of amputation or fracture. CONCLUSIONS In patients with type 2 diabetes and kidney disease, the risk of kidney failure and cardiovascular events was lower in the canagliflozin group than in the placebo group at a median follow-up of 2.62 years

Minimal information for studies of extracellular vesicles (MISEV2023): From basic to advanced approaches

Extracellular vesicles (EVs), through their complex cargo, can reflect the state of their cell of origin and change the functions and phenotypes of other cells. These features indicate strong biomarker and therapeutic potential and have generated broad interest, as evidenced by the steady year-on-year increase in the numbers of scientific publications about EVs. Important advances have been made in EV metrology and in understanding and applying EV biology. However, hurdles remain to realising the potential of EVs in domains ranging from basic biology to clinical applications due to challenges in EV nomenclature, separation from non-vesicular extracellular particles, characterisation and functional studies. To address the challenges and opportunities in this rapidly evolving field, the International Society for Extracellular Vesicles (ISEV) updates its 'Minimal Information for Studies of Extracellular Vesicles', which was first published in 2014 and then in 2018 as MISEV2014 and MISEV2018, respectively. The goal of the current document, MISEV2023, is to provide researchers with an updated snapshot of available approaches and their advantages and limitations for production, separation and characterisation of EVs from multiple sources, including cell culture, body fluids and solid tissues. In addition to presenting the latest state of the art in basic principles of EV research, this document also covers advanced techniques and approaches that are currently expanding the boundaries of the field. MISEV2023 also includes new sections on EV release and uptake and a brief discussion of in vivo approaches to study EVs. Compiling feedback from ISEV expert task forces and more than 1000 researchers, this document conveys the current state of EV research to facilitate robust scientific discoveries and move the field forward even more rapidly

Omecamtiv mecarbil in chronic heart failure with reduced ejection fraction, GALACTIC‐HF: baseline characteristics and comparison with contemporary clinical trials

Aims: The safety and efficacy of the novel selective cardiac myosin activator, omecamtiv mecarbil, in patients with heart failure with reduced ejection fraction (HFrEF) is tested in the Global Approach to Lowering Adverse Cardiac outcomes Through Improving Contractility in Heart Failure (GALACTIC‐HF) trial. Here we describe the baseline characteristics of participants in GALACTIC‐HF and how these compare with other contemporary trials. Methods and Results: Adults with established HFrEF, New York Heart Association functional class (NYHA) ≥ II, EF ≤35%, elevated natriuretic peptides and either current hospitalization for HF or history of hospitalization/ emergency department visit for HF within a year were randomized to either placebo or omecamtiv mecarbil (pharmacokinetic‐guided dosing: 25, 37.5 or 50 mg bid). 8256 patients [male (79%), non‐white (22%), mean age 65 years] were enrolled with a mean EF 27%, ischemic etiology in 54%, NYHA II 53% and III/IV 47%, and median NT‐proBNP 1971 pg/mL. HF therapies at baseline were among the most effectively employed in contemporary HF trials. GALACTIC‐HF randomized patients representative of recent HF registries and trials with substantial numbers of patients also having characteristics understudied in previous trials including more from North America (n = 1386), enrolled as inpatients (n = 2084), systolic blood pressure < 100 mmHg (n = 1127), estimated glomerular filtration rate < 30 mL/min/1.73 m2 (n = 528), and treated with sacubitril‐valsartan at baseline (n = 1594). Conclusions: GALACTIC‐HF enrolled a well‐treated, high‐risk population from both inpatient and outpatient settings, which will provide a definitive evaluation of the efficacy and safety of this novel therapy, as well as informing its potential future implementation

Direct assessment and validation of allele specific transcription factor binding in the human genome

Characterization of human genetic variation has focused on expression quantitative trait loci (eQTL) mapping; however, direct assessment of cis-regulatory variation requires allele-specific approaches. Measuring allelic expression (AE) on a genome-wide scale appears more powerful as environmental and trans-acting influences are minimized. Results indicate that allele-specific differences in transcript expression within an individual can affect up to 30% of loci. The underlying variants can be identified by mapping differences in AE on Illumina BeadChips. Over 50% of population variance in AE is explained by mapped cis-rSNPs. Studies show that these cis-rSNPs have been implicated in differences in transcription factor (TF) binding, suggesting that TF action can be further investigated using population variation as a tool. In this thesis, these approaches have been extended to explore allele-specific TF binding using the model NF-κB by monitoring the consequences of gene knockdown in a genome-wide manner. NF-κB has been shown to be involved in the immune response and the NF-κB motif is enriched in lymphoblastoid cell lines (LCLs), mainly in promoters and strong enhancer elements. We intersected mapped candidate cis-rSNPs detected in LCLs in our above experiments as well as matched control SNPs from HapMap YRI and CEU populations with publicly available NF-κB Chromatin Immunoprecipitation (ChIP)-seq experiments from the ENCODE project. Preliminary analysis of regions surrounding candidate cis-rSNPs were enriched in NF-κB binding sites versus matched controls, with 39.0 % of top SNPs overlapping at least one NF-κB ChIP-seq peak. To elucidate the impact of candidate SNPs on AE imbalances, we performed TNF- α induction coupled to inhibition of NF-κB in LCLs followed by AE analysis on Illumina HumanOmni5-Quad BeadChips. We used in house mapped cis-regulatory variants in the LCL population merged with data from the aforementioned experiment. Our data set, which consisted of loci associated to top 10 cis-rSNPs ranked by p-value (pv; top10= 10 most significant p-values) that showed diminished AE upon perturbation of NF-κB were overlapped with publicly available data. This data consisted of ENCODE ChIP-seq peaks and TRANSFAC binding sites for NF-κB and known cooperative TFs of NF-κB. Loci that had an AE change at greater than 3 SNPs upon perturbation of NF-κB and were associated to top heterozygous SNPs (rank 1, 2, 3) yielded 581 cases out of ~1700. Analysis of top 3 ¬cis-rSNPs described showed a significant difference of over 5-fold between case and control SNPs, such that 64% of loci had a top 3 heterozygous SNP that was found in an LCL specific TF ChIP-seq peak or TRANSFAC binding site for NF-κB or a known cooperative TF of NF-κB. Bioinformatics analysis suggests that identified SNPs are essential for NF-κB binding. A case study was also done in order to perturb the TF SNAI1 because we had a strong hypothesis for the association of SNAI1 and WNT4, as well as, evidence for its role in fibroblasts (FBs). We were not able to reproduce the effect of SNAI1 on WNT4 in vivo. Upon comparison of the regulatory role of NF-κB and SNAI1 in LCLs and FBs, respectively; we observed that NF-κB had a regulatory effect on approximately 33% of loci in comparison to only approximately 2% of loci for SNAI1 in FBs. This study illustrates that key regulatory TFs, such as NF-κB in LCLs, can be globally studied at a single base resolution in living cells using a combination of perturbation and sensitive measurements with allelic resolution.La caractérisation de la variation génétique a mis l'accent sur les loci d'expression de caractères quantitatifs(eLCQ); cependant, l'évaluation directe des variations régulatrices en cis nécessite des approches allèle-spécifique (cis-rSNPs). La mesure de l'expression allélique (EA) est très efficace puisque les perturbations environnementales et les influences en trans sont réduites. Les résultats indiquent que les différences d'EA peuvent affecter jusqu'à 30% des loci chez un individu. Les polymorphismes responsable de telles variations peuvent être identifiés par cartographie des différences d'EA en utilisant des puces de génotypage Illumina. Ainsi, plus de 50% de la variance en EA de la population est expliqué par la cartographie des cis-rSNPs. Des études ont montré que ces cis-rSNPs ont été impliqués dans des différences de liason de facteurs de transcription (FT). Celà suggère que l'étude du mode d'action de ces FT pourrait être approfondie par l'utilisation comme outil des polymorphismes dans la population. Nous avons appliqué cette approche au FT NF-κB et analysé les conséquences de l'inactivation de ce gène à l'échelle du genome. Des données récentes montrent l'implication de NF-κB dans la réponse immunitaire et son motif de liaison à l'ADN est retrouvé enrichi dans les cellules lymphoblastoïdes humaines (LCL), surtout au niveau des promoteurs et des activateurs transcriptionnels. Nous avons croisé les cis-rSNPs cartographiés dans des LCLs des populations HapMap YRI et CEU, ainsi que des SNPs de contrôles, avec des données d'immunoprécipitation de chromatine suivi de séquençage à haut débit (ChIP-seq) utilisant l'anticorps NF-κB du projet ENCODE et accessible au public. Des analyses préliminaires des régions contenant les cis-rSNPs candidats ont montré un enrichissement des sites de liaison pour le facteur NF-κB par rapport aux sites contrôles. En effet, 39% des sites candidats sont situés dans un site de liaison pour NF-kB. Afin d'étudier le rôle potentiel des cis-rSNPs sur l'EA différentielle, nous avons réalisé des expériences d'induction de TNF-α couplé à l'inhibition de NF-κB dans les LCLs suivie par l'analyses de l'EA sur des puces de génotypage Illumina 5M. Nous avons ensuite comparé ces données avec la cartographie de cis-rSNPs dans des LCLs générée dans notre laboratoire.Nos données sont composées de cis-rSNPs associés à l'EA différentielle de loci suite à la perturbation de NF-κB et classés par valeur p (pv; top10= les 10 valeurs les plus significatives) et ont été croisées avec des données accessibles au public. Ces données sont composées des coordonnées de pics de ChIP-seq et des sites de liaison TRANSFAC pour le facteur NF-κB et de ses co-régulateurs transcriptionnels connus. Les loci montrant des différences d'EA suite à la perturbation de NF-κB et avec 1 ou plusieurs des cis-rSNPs (« top3 » pv) hétérozygotes dans les individus étudiés étaient aux nombre de 581. La recherche de ces cis-rSNPs « top 3 » dans les sites de liaison (pics de ChIP-seq) dans les LCLs ou dans un site de TRANSFAC pour NF-κB ou pour un de ses co-régulateurs transcriptionnels montrent un enrichissement significatif (64% des loci) avec un ratio supérieur à 5 par rapport aux SNPs de contrôles.L'analyse bioinformatique suggère que les SNPs identifiés sont essentiels pour la liason de NF-κB. Une étude complémentaire à consisté à perturber le FT SNAI1 probablement associé au gène WNT4 et présentant un rôle important dans les fibroblastes (FB). Cependant, nous n'avons pas été en mesure de reproduire l'effet de SNAI1 sur WNT4 in vivo.Nous avons ensuite comparé les rôles régulateurs de NF-κB et de SNAI1 dans les LCLs et les FBs respectivement (par inactivation de ces gènes). Nous avons observé que NF-κB a un effet régulateurs sur environ 33% des loci dans les LCLs contre seulement 2 % pour SNAI1 dans des FBs.Cette étude supporte l'utilisation de perturbations de l'expression de FT pour étudier le rôle clé de FTs régulateurs dans un type cellulaire donné

Effect of SGLT2 Inhibitors on Stroke and Atrial Fibrillation in Diabetic Kidney Disease: Results From the CREDENCE Trial and Meta-Analysis

BACKGROUND AND PURPOSE: Chronic kidney disease with reduced estimated glomerular filtration rate or elevated albuminuria increases risk for ischemic and hemorrhagic stroke. This study assessed the effects of sodium glucose cotransporter 2 inhibitors (SGLT2i) on stroke and atrial fibrillation/flutter (AF/AFL) from CREDENCE (Canagliflozin and Renal Events in Diabetes With Established Nephropathy Clinical Evaluation) and a meta-analysis of large cardiovascular outcome trials (CVOTs) of SGLT2i in type 2 diabetes mellitus.METHODS: CREDENCE randomized 4401 participants with type 2 diabetes mellitus and chronic kidney disease to canagliflozin or placebo. Post hoc, we estimated effects on fatal or nonfatal stroke, stroke subtypes, and intermediate markers of stroke risk including AF/AFL. Stroke and AF/AFL data from 3 other completed large CVOTs and CREDENCE were pooled using random-effects meta-analysis.RESULTS: In CREDENCE, 142 participants experienced a stroke during follow-up (10.9/1000 patient-years with canagliflozin, 14.2/1000 patient-years with placebo; hazard ratio [HR], 0.77 [95% CI, 0.55-1.08]). Effects by stroke subtypes were: ischemic (HR, 0.88 [95% CI, 0.61-1.28]; n=111), hemorrhagic (HR, 0.50 [95% CI, 0.19-1.32]; n=18), and undetermined (HR, 0.54 [95% CI, 0.20-1.46]; n=17). There was no clear effect on AF/AFL (HR, 0.76 [95% CI, 0.53-1.10]; n=115). The overall effects in the 4 CVOTs combined were: total stroke (HRpooled, 0.96 [95% CI, 0.82-1.12]), ischemic stroke (HRpooled, 1.01 [95% CI, 0.89-1.14]), hemorrhagic stroke (HRpooled, 0.50 [95% CI, 0.30-0.83]), undetermined stroke (HRpooled, 0.86 [95% CI, 0.49-1.51]), and AF/AFL (HRpooled, 0.81 [95% CI, 0.71-0.93]). There was evidence that SGLT2i effects on total stroke varied by baseline estimated glomerular filtration rate (P=0.01), with protection in the lowest estimated glomerular filtration rate (<45 mL/min/1.73 m2]) subgroup (HRpooled, 0.50 [95% CI, 0.31-0.79]).CONCLUSIONS: Although we found no clear effect of SGLT2i on total stroke in CREDENCE or across trials combined, there was some evidence of benefit in preventing hemorrhagic stroke and AF/AFL, as well as total stroke for those with lowest estimated glomerular filtration rate. Future research should focus on confirming these data and exploring potential mechanisms. Registration: URL: https://www.clinicaltrials.gov; Unique identifier: NCT02065791