Unraveling the effect of silent, intronic and missense mutations on VWF splicing: contribution of next generation sequencing in the study of mRNA

Large studies in von Willebrand disease patients, including Spanish and Portuguese registries, led to the identification of >250 different mutations. It is a challenge to determine the pathogenic effect of potential splice site mutations on VWF mRNA. This study aimed to elucidate the true effects of 18 mutations on VWF mRNA processing, investigate the contribution of next-generation sequencing to in vivo mRNA study in von Willebrand disease, and compare the findings with in silico prediction. RNA extracted from patient platelets and leukocytes was amplified by RT-PCR and sequenced using Sanger and next generation sequencing techniques. Eight mutations affected VWF splicing: c.1533+1G>A, c.5664+2T>C and c.546G>A (p.=) prompted exon skipping; c.3223-7_3236dup and c.7082-2A>G resulted in activation of cryptic sites; c.3379+1G>A and c.7437G>A) demonstrated both molecular pathogenic mechanisms simultaneously; and the p.Cys370Tyr missense mutation generated two aberrant transcripts. Of note, the complete effect of three mutations was provided by next generation sequencing alone because of low expression of the aberrant transcripts. In the remaining 10 mutations, no effect was elucidated in the experiments. However, the differential findings obtained in platelets and leukocytes provided substantial evidence that four of these would have an effect on VWF levels. In this first report using next generation sequencing technology to unravel the effects of VWF mutations on splicing, the technique yielded valuable information. Our data bring to light the importance of studying the effect of synonymous and missense mutations on VWF splicing to improve the current knowledge of the molecular mechanisms behind von Willebrand disease. clinicaltrials.gov identifier:02869074

Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)1.

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field

Zolmitriptan: A Novel Portal Hypotensive Agent Which Synergizes with Propranolol in Lowering Portal Pressure

Objective: Only a limited proportion of patients needing pharmacological control of portal hypertension are hemodynamic responders to propranolol. Here we analyzed the effects of zolmitriptan on portal pressure and its potential interaction with propranolol. Methods: Zolmitriptan, propranolol or both were tested in two rat models of portal hypertension: common bile duct ligation (CBDL) and CCl4-induced cirrhosis. In these animals we measured different hemodynamic parameters including portal venous pressure, arterial renal flow, portal blood flow and cardiac output. We also studied the changes in superior mesenteric artery perfusion pressure and in arterial wall cAMP levels induced by zolmitriptan, propranolol or both. Moreover, we determined the effect of splanchnic sympathectomy on the response of PVP to zolmitriptan. Results: In both models of portal hypertension zolmitriptan induced a dose-dependent transient descent of portal pressure accompanied by reduction of portal flow with only slight decrease in renal flow. In cirrhotic rats, splanchnic sympathectomy intensified and prolonged zolmitriptan-induced portal pressure descent. Also, propranolol caused more intense and durable portal pressure fall when combined with zolmitriptan. Mesenteric artery perfusion pressure peaked for about 1 min upon zolmitriptan administration but showed no change with propranolol. However propranolol enhanced and prolonged the elevation in mesenteric artery perfusion pressure induced by zolmitriptan. In vitro studies showed that propranolol prevented the inhibitory effects of b2-agonists on zolmitriptan-induced vasoconstriction and the combination of propranolol and zolmitriptan significantly reduced the elevation of cAMP caused by b2-agonists. Conclusion: Zolmitriptan reduces portal hypertension and non-selective beta-blockers can improve this effect. Combinati

Primary constitutional MLH1 epimutations: a focal epigenetic event

BACKGROUND: Constitutional MLH1 epimutations are characterised by monoallelic methylation of the MLH1 promoter throughout normal tissues, accompanied by allele-specific silencing. The mechanism underlying primary MLH1 epimutations is currently unknown. The aim of this study was to perform an in-depth characterisation of constitutional MLH1 epimutations targeting the aberrantly methylated region around MLH1 and other genomic loci. METHODS: Twelve MLH1 epimutation carriers, 61 Lynch syndrome patients, and 41 healthy controls, were analysed by Infinium 450 K array. Targeted molecular techniques were used to characterise the MLH1 epimutation carriers and their inheritance pattern. RESULTS: No nucleotide or structural variants were identified in-cis on the epimutated allele in 10 carriers, in which inter-generational methylation erasure was demonstrated in two, suggesting primary type of epimutation. CNVs outside the MLH1 locus were found in two cases. EPM2AIP1-MLH1 CpG island was identified as the sole differentially methylated region in MLH1 epimutation carriers compared to controls. CONCLUSION: Primary constitutional MLH1 epimutations arise as a focal epigenetic event at the EPM2AIP1-MLH1 CpG island in the absence of cis-acting genetic variants. Further molecular characterisation is needed to elucidate the mechanistic basis of MLH1 epimutations and their heritability/reversibility

Procalcitonina es superior a recuento linfocitario, índice neutrófilo/linfocito y proteína C reactiva para la predicción de mortalidad a 30 días de pacientes con COVID-19 en el servicio de urgencias = Procalcitonin concentration in the emergency department predicts 30-day mortality in COVID-19 better than the lymphocyte count, the neutrophil-tolymphocyte ratio, or the C-reactive protein level.

Although many demographic and clinical predictors of mortality have been studied in relation to COVID-19, little has been reported about the prognostic utility of inflammatory biomarkers. Retrospective cohort study. All patients with laboratory-confirmed COVID-19 treated in a hospital emergency department were included consecutively if baseline measurements of the following biomarkers were on record: lymphocyte counts, neutrophil-to-lymphocyte ratio NRL, and C-reactive protein (CRP) and procalcitonin (PCT) levels. We analyzed associations between the biomarkers and all-cause 30-day mortality using Cox regression models and dose-response curves. We included 896 patients, 151 (17%) of whom died within 30 days. The median (interquartile range) age was 63 (51-78) years, and 494 (55%) were men. NLR, CRP and PCT levels at ED presentation were higher, while lymphocyte counts were lower, in patients who died compared to those who survived (P .001). The areas under the receiver operating characteristic curves revealed the PCT concentration (0.79; 95% CI, 0.75-0.83) to be a better predictor of 30-day mortality than the lymphocyte count (0.70; 95% CI, 0.65-0.74; P .001), the NLR (0.74; 95% CI, 0.69-0.78; P = .03), or the CRP level (0.72; 95% CI, 0.68-0.76; P .001). The proposed PCT concentration decision points for use in emergency department case management were 0.06 ng/L (negative) and 0.72 ng/L (positive). These cutoffs helped classify risk in 357 patients (40%). Multivariable analysis demonstrated that the PCT concentration had the strongest association with mortality. PCT concentration in the emergency department predicts all-cause 30-day mortality in patients with COVID-19 better than other inflammatory biomarkers