A community-based resource for automatic exome variant-calling and annotation in Mendelian disorders

BACKGROUND: Mendelian disorders are mostly caused by single mutations in the DNA sequence of a gene, leading to a phenotype with pathologic consequences. Whole Exome Sequencing of patients can be a cost-effective alternative to standard genetic screenings to find causative mutations of genetic diseases, especially when the number of cases is limited. Analyzing exome sequencing data requires specific expertise, high computational resources and a reference variant database to identify pathogenic variants. RESULTS: We developed a database of variations collected from patients with Mendelian disorders, which is automatically populated thanks to an associated exome-sequencing pipeline. The pipeline is able to automatically identify, annotate and store insertions, deletions and mutations in the database. The resource is freely available online http://exome.tigem.it. The exome sequencing pipeline automates the analysis workflow (quality control and read trimming, mapping on reference genome, post-alignment processing, variation calling and annotation) using state-of-the-art software tools. The exome-sequencing pipeline has been designed to run on a computing cluster in order to analyse several samples simultaneously. The detected variants are annotated by the pipeline not only with the standard variant annotations (e.g. allele frequency in the general population, the predicted effect on gene product activity, etc.) but, more importantly, with allele frequencies across samples progressively collected in the database itself, stratified by Mendelian disorder. CONCLUSIONS: We aim at providing a resource for the genetic disease community to automatically analyse whole exome-sequencing samples with a standard and uniform analysis pipeline, thus collecting variant allele frequencies by disorder. This resource may become a valuable tool to help dissecting the genotype underlying the disease phenotype through an improved selection of putative patient-specific causative or phenotype-associated variations

Mantra 2.0: an online collaborative resource for drug mode of action and repurposing by network analysis

Abstract Summary: Elucidation of molecular targets of a compound [mode of action (MoA)] and its off-targets is a crucial step in drug development. We developed an online collaborative resource (MANTRA 2.0) that supports this process by exploiting similarities between drug-induced transcriptional profiles. Drugs are organized in a network of nodes (drugs) and edges (similarities) highlighting 'communities' of drugs sharing a similar MoA. A user can upload gene expression profiles before and after drug treatment in one or multiple cell types. An automated processing pipeline transforms the gene expression profiles into a unique drug 'node' embedded in the drug-network. Visual inspection of the neighbouring drugs and communities helps in revealing its MoA and to suggest new applications of known drugs (drug repurposing). MANTRA 2.0 allows storing and sharing user-generated network nodes, thus making MANTRA 2.0 a collaborative ever-growing resource. Availability and implementation: The web tool is freely available for academic use at http://mantra.tigem.it. Contact: [email protected]

Mantra 2.0: An online collaborative resource for drug mode of action and repurposing by network analysis

Elucidation of molecular targets of a compound (mode of action, MoA) and of its off-targets is a crucial step in drug development. We developed an online collaborative resource (MANTRA 2.0) that supports this process by exploiting similarities between drug-induced transcriptional profiles. Drugs are organised in a network of nodes (drugs) and edges (similarities) highlighting “communities” of drugs sharing a similar MoA. A user can upload gene expression profiles (GEPs) before and after drug treatment in one or multiple cell types. An automated processing pipeline transforms the GEPs into a unique drug ”node” embedded in the drug-network. Visual inspection of the neighbouring drugs and communities helps in revealing its MoA, and to suggest new applications of known drugs (drug repurposing). MANTRA 2.0 allows storing and sharing user-generated network nodes, thus making MANTRA 2.0 a collaborative ever-growing resource

Blood stem cell-forming haemogenic endothelium in zebrafish derives from arterial endothelium

Haematopoietic stem cells are generated from the haemogenic endothelium (HE) located in the floor of the dorsal aorta (DA). Despite being integral to arteries, it is controversial whether HE and arterial endothelium share a common lineage. Here, we present a transgenic zebrafish runx1 reporter line to isolate HE and aortic roof endothelium (ARE)s, excluding non-aortic endothelium. Transcriptomic analysis of these populations identifies Runx1-regulated genes and shows that HE initially expresses arterial markers at similar levels to ARE. Furthermore, runx1 expression depends on prior arterial programming by the Notch ligand dll4. Runx1andminus;/andminus; mutants fail to downregulate arterial genes in the HE, which remains integrated within the DA, suggesting that Runx1 represses the pre-existing arterial programme in HE to allow progression towards the haematopoietic fate. These findings strongly suggest that, in zebrafish, aortic endothelium is a precursor to HE, with potential implications for pluripotent stem cell differentiation protocols for the generation of transplantable HSCs.</p

Deletion of a conserved Gata2 enhancer impairs haemogenic endothelium programming and adult Zebrafish haematopoiesis

Gata2 is a key transcription factor required to generate Haematopoietic Stem and Progenitor Cells (HSPCs) from haemogenic endothelium (HE); misexpression of Gata2 leads to haematopoietic disorders. Here we deleted a conserved enhancer (i4 enhancer) driving pan-endothelial expression of the zebrafish gata2a and showed that Gata2a is required for HE programming by regulating expression of runx1 and of the second Gata2 orthologue, gata2b. By 5 days, homozygous gata2aΔi4/Δi4 larvae showed normal numbers of HSPCs, a recovery mediated by Notch signalling driving gata2b and runx1 expression in HE. However, gata2aΔi4/Δi4 adults showed oedema, susceptibility to infections and marrow hypo-cellularity, consistent with bone marrow failure found in GATA2 deficiency syndromes. Thus, gata2a expression driven by the i4 enhancer is required for correct HE programming in embryos and maintenance of steady-state haematopoietic stem cell output in the adult. These enhancer mutants will be useful in exploring further the pathophysiology of GATA2-related deficiencies in vivo

Etv6 activates vegfa expression through positive and negative transcriptional regulatory networks in Xenopus embryos

How vegfa expression is transcriptionally regulated in vivo is unclear. Here, the authors demonstrate that the ETS transcription factor Etv6 acts as a repressor and an activator of two direct regulators of vegfa expression (foxo3 and klf4, respectively) to control blood formation in Xenopus

Major thoracic surgery in Jehovah's witness: A multidisciplinary approach case report

Introduction: A bloodless surgery can be desirable also for non Jehovah’s witnesses patients, but requires a team approach from the very first assessment to ensure adequate planning. Presentation of the case: Our patient, a Jehovah’s witnesses, was scheduled for right lower lobectomy due to pulmonary adenocarcinoma. Her firm denies to receive any kind of transfusions, forced clinicians to a bloodless management of the case. Discussion: Before surgery a meticulous coagulopathy research and hemodynamic optimization are useful to prepare patient to operation. During surgery, controlled hypotension can help to obtain effective hemostasis. After surgery, clinicians monitored any possible active bleeding, using continuous noninvasive hemoglobin monitoring, limiting the blood loss due to serial in vitro testing. The optimization of cardiac index and delivery of oxygen were continued to grant a fast recovery. Conclusion: Bloodless surgery is likely to gain popularity, and become standard practice for all patients. The need for transfusion should be targeted on individual case, avoiding strictly fixed limit often leading to unnecessary transfusion

Choroidal Changes in Blood Flow in Patients with Intermediate AMD after Oral Dietary Supplement Based on Astaxanthin, Bromelain, Vitamin D3, Folic Acid, Lutein, and Antioxidants

Background and Objectives: The aim of this study was to investigate the impact of oral administration of the combination of astaxanthin (AXT), lutein, folic acid, vitamin D3, and bromelain with antioxidants on choroidal blood flow in patients with age-related intermediate macular degeneration (AMD). Materials and Methods: Patients affected by intermediate AMD and treated with daily oral nutritional supplement with AXT, bromelain, vitamin D3, folic acid, lutein, and antioxidants for a period of at least 6 months were included in this retrospective study. A control group homogenous for age and sex was also included in the analysis. All participants underwent a complete ophthalmologic examination, spectral domain optical coherence tomography (SD-OCT), and optical coherence tomography angiography (OCTA) evaluation. Outcome measures were choroidal thickness (CHT) and choriocapillary vessel density (CCVD) after six months of AXT assumption. Results: CCVD values showed statistically significant difference between cases and controls at baseline (p &lt; 0.001) and in the cases during follow-up (p &lt; 0.001). The CHT measurements showed statistically significant difference between cases and controls (p = 0.002) and in the cases during follow-up (p &lt; 0.001). Conclusions: The combined use of structural OCT and OCTA allows for a detailed analysis in vivo of perfusion parameters of the choriocapillaris and choroid and evaluation of changes of choroidal blood flow after oral nutritional supplements that affect blood flow velocity