Network-Based Methods for Approaching Human Pathologies from a Phenotypic Point of View

Network and systemic approaches to studying human pathologies are helping us to gain insight into the molecular mechanisms of and potential therapeutic interventions for human diseases, especially for complex diseases where large numbers of genes are involved. The complex human pathological landscape is traditionally partitioned into discrete “diseases”; however, that partition is sometimes problematic, as diseases are highly heterogeneous and can differ greatly from one patient to another. Moreover, for many pathological states, the set of symptoms (phenotypes) manifested by the patient is not enough to diagnose a particular disease. On the contrary, phenotypes, by definition, are directly observable and can be closer to the molecular basis of the pathology. These clinical phenotypes are also important for personalised medicine, as they can help stratify patients and design personalised interventions. For these reasons, network and systemic approaches to pathologies are gradually incorporating phenotypic information. This review covers the current landscape of phenotype-centred network approaches to study different aspects of human diseasesThis work was partially funded by The Spanish Ministry of Economy and Competitiveness with European Regional Development Fund [grant numbers PID2019-108096RB-C21 and PID2019-108096RB-C22]; the European Food Safety Authority [grant number GP/EFSA/ENCO/2020/02]; the Andalusian Government with European Regional Development Fund [grant numbers UMA18- FEDERJA-102 and PAIDI 2020:PY20-00372]; Fundacion Progreso y Salud [grant number PI-0075-2017], also from the Andalusian Government; the Ramón Areces foundation, which funds project for the investigation of rare disease (National call for research on life and material sciences, XIX edition); the University of Malaga (Ayudas del I Plan Propio) and the Institute of Health Carlos III which funds the IMPaCT-Data project. The CIBERER is an initiative from the Institute of Health Carlos III. The conclusions, findings and opinions expressed in this scientific paper reflect only the view of the authors and not the official position of the European Food Safety Authority. Partial funding for open access charge: Universidad de Málag

Artificial Neural Network Inference (ANNI): A Study on Gene-Gene Interaction for Biomarkers in Childhood Sarcomas

Objective: To model the potential interaction between previously identified biomarkers in children sarcomas using artificial neural network inference (ANNI). Method: To concisely demonstrate the biological interactions between correlated genes in an interaction network map, only 2 types of sarcomas in the children small round blue cell tumors (SRBCTs) dataset are discussed in this paper. A backpropagation neural network was used to model the potential interaction between genes. The prediction weights and signal directions were used to model the strengths of the interaction signals and the direction of the interaction link between genes. The ANN model was validated using Monte Carlo cross-validation to minimize the risk of over-fitting and to optimize generalization ability of the model. Results: Strong connection links on certain genes (TNNT1 and FNDC5 in rhabdomyosarcoma (RMS); FCGRT and OLFM1 in Ewing’s sarcoma (EWS)) suggested their potency as central hubs in the interconnection of genes with different functionalities. The results showed that the RMS patients in this dataset are likely to be congenital and at low risk of cardiomyopathy development. The EWS patients are likely to be complicated by EWS-FLI fusion and deficiency in various signaling pathways, including Wnt, Fas/Rho and intracellular oxygen. Conclusions: The ANN network inference approach and the examination of identified genes in the published literature within the context of the disease highlights the substantial influence of certain genes in sarcomas

The Comprehensive Native Interactome of a Fully Functional Tagged Prion Protein

The enumeration of the interaction partners of the cellular prion protein, PrPC, may help clarifying its elusive molecular function. Here we added a carboxy proximal myc epitope tag to PrPC. When expressed in transgenic mice, PrPmyc carried a GPI anchor, was targeted to lipid rafts, and was glycosylated similarly to PrPC. PrPmyc antagonized the toxicity of truncated PrP, restored prion infectibility of PrPC-deficient mice, and was physically incorporated into PrPSc aggregates, indicating that it possessed all functional characteristics of genuine PrPC. We then immunopurified myc epitope-containing protein complexes from PrPmyc transgenic mouse brains. Gentle differential elution with epitope-mimetic decapeptides, or a scrambled version thereof, yielded 96 specifically released proteins. Quantitative mass spectrometry with isotope-coded tags identified seven proteins which co-eluted equimolarly with PrPC and may represent component of a multiprotein complex. Selected PrPC interactors were validated using independent methods. Several of these proteins appear to exert functions in axomyelinic maintenance

Identificación de módulos asociados a fenotipos patológicos

Trabajo fin de máster en Bioinformática y Biología ComputacionalCopy Number Variations (CNVs) are genomic structural variations frequently observed in healthy individuals, but can also lead to disease. They are the etiological cause of many rare genomic disorders that affect a large number of people in population, constituting a major public health problem. Unlike other small mutations, deleterious CNVs can reach millions of nucleotides containing several genes and other functional DNA regions. Many of these CNVs have yet unknown relationships to the phenotypes observed in patients. Therefore, the identification of the potentially affected molecular and genetical mechanisms in the CNVs and their relation with certain phenotypes in patients with rare deleterious disorders, nowadays, remains as a big challenge for clinical geneticists. Based on different datasets that links phenotypes, patients and genomic loci, two systemic approaches were used to understand the molecular basis that underlie those CNVs. Firstly, a functional analysis of the genes coded in these regions is carried out to realise which are the biological processes affected by the CNVs mutations thus to the phenotypes. Secondly, a network propagation analysis is done to expand the knowledge of the query genes and its interactome context. The results obtained for a cluster of patients and a number of phenotypes of clinical interest are briefly explaine

A Knowledge-based Integrative Modeling Approach for <em>In-Silico</em> Identification of Mechanistic Targets in Neurodegeneration with Focus on Alzheimer’s Disease

Dementia is the progressive decline in cognitive function due to damage or disease in the body beyond what might be expected from normal aging. Based on neuropathological and clinical criteria, dementia includes a spectrum of diseases, namely Alzheimer's dementia, Parkinson's dementia, Lewy Body disease, Alzheimer's dementia with Parkinson's, Pick's disease, Semantic dementia, and large and small vessel disease. It is thought that these disorders result from a combination of genetic and environmental risk factors. Despite accumulating knowledge that has been gained about pathophysiological and clinical characteristics of the disease, no coherent and integrative picture of molecular mechanisms underlying neurodegeneration in Alzheimer’s disease is available. Existing drugs only offer symptomatic relief to the patients and lack any efficient disease-modifying effects. The present research proposes a knowledge-based rationale towards integrative modeling of disease mechanism for identifying potential candidate targets and biomarkers in Alzheimer’s disease. Integrative disease modeling is an emerging knowledge-based paradigm in translational research that exploits the power of computational methods to collect, store, integrate, model and interpret accumulated disease information across different biological scales from molecules to phenotypes. It prepares the ground for transitioning from ‘descriptive’ to “mechanistic” representation of disease processes. The proposed approach was used to introduce an integrative framework, which integrates, on one hand, extracted knowledge from the literature using semantically supported text-mining technologies and, on the other hand, primary experimental data such as gene/protein expression or imaging readouts. The aim of such a hybrid integrative modeling approach was not only to provide a consolidated systems view on the disease mechanism as a whole but also to increase specificity and sensitivity of the mechanistic model by providing disease-specific context. This approach was successfully used for correlating clinical manifestations of the disease to their corresponding molecular events and led to the identification and modeling of three important mechanistic components underlying Alzheimer’s dementia, namely the CNS, the immune system and the endocrine components. These models were validated using a novel in-silico validation method, namely biomarker-guided pathway analysis and a pathway-based target identification approach was introduced, which resulted in the identification of the MAPK signaling pathway as a potential candidate target at the crossroad of the triad components underlying disease mechanism in Alzheimer’s dementia

Distinct translatome changes in specific neural populations precede electroencephalographic changes in prion-infected mice

Selective vulnerability is an enigmatic feature of neurodegenerative diseases (NDs), whereby a widely expressed protein causes lesions in specific cell types and brain regions. Using the RiboTag method in mice, translational responses of five neural subtypes to acquired prion disease (PrD) were measured. Pre-onset and disease onset timepoints were chosen based on longitudinal electroencephalography (EEG) that revealed a gradual increase in theta power between 10- and 18-weeks after prion injection, resembling a clinical feature of human PrD. At disease onset, marked by significantly increased theta power and histopathological lesions, mice had pronounced translatome changes in all five cell types despite appearing normal. Remarkably, at a pre-onset stage, prior to EEG and neuropathological changes, we found that 1) translatomes of astrocytes indicated reduced synthesis of ribosomal and mitochondrial components, 2) glutamatergic neurons showed increased expression of cytoskeletal genes, and 3) GABAergic neurons revealed reduced expression of circadian rhythm genes. These data demonstrate that early translatome responses to neurodegeneration emerge prior to conventional markers of disease and are cell type-specific. Therapeutic strategies may need to target multiple pathways in specific populations of cells, early in disease

Vacuolar ATPase Is a Possible Therapeutic Target in Acute Myeloid Leukemia: Focus on Patient Heterogeneity and Treatment Toxicity

Vacuolar ATPase (V-ATPase) is regarded as a possible target in cancer treatment. It is expressed in primary acute myeloid leukemia cells (AML), but the expression varies between patients and is highest for patients with a favorable prognosis after intensive chemotherapy. We therefore investigated the functional effects of two V-ATPase inhibitors (bafilomycin A1, concanamycin A) for primary AML cells derived from 80 consecutive patients. The V-ATPase inhibitors showed dose-dependent antiproliferative and proapoptotic effects that varied considerably between patients. A proteomic comparison of primary AML cells showing weak versus strong antiproliferative effects of V-ATPase inhibition showed a differential expression of proteins involved in intracellular transport/cytoskeleton functions, and an equivalent phosphoproteomic comparison showed a differential expression of proteins that regulate RNA processing/function together with increased activity of casein kinase 2. Patients with secondary AML, i.e., a heterogeneous subset with generally adverse prognosis and previous cytotoxic therapy, myeloproliferative neoplasia or myelodysplastic syndrome, were characterized by a strong antiproliferative effect of V-ATPase inhibition and also by a specific mRNA expression profile of V-ATPase interactome proteins. Furthermore, the V-ATPase inhibition altered the constitutive extracellular release of several soluble mediators (e.g., chemokines, interleukins, proteases, protease inhibitors), and increased mediator levels in the presence of AML-supporting bone marrow mesenchymal stem cells was then observed, especially for patients with secondary AML. Finally, animal studies suggested that the V-ATPase inhibitor bafilomycin had limited toxicity, even when combined with cytarabine. To conclude, V-ATPase inhibition has antileukemic effects in AML, but this effect varies between patients.publishedVersio

Vacuolar ATPase Is a Possible Therapeutic Target in Acute Myeloid Leukemia: Focus on Patient Heterogeneity and Treatment Toxicity

This work was supported by Kreftforeningen, the Norwegian Cancer Society (grant no. 100933). The Genomics Core Facility (GCF) is supported in part by major grants from the Research Council of Norway (grant no. 245979/F50) and Bergen Research Foundation. The GCF at the University of Bergen, which is part of the NorSeq consortium, provided support in ChIP-Seq bioinformatics analysis.Supplementary Materials: The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/jcm12175546/s1Vacuolar ATPase (V-ATPase) is regarded as a possible target in cancer treatment. It is expressed in primary acute myeloid leukemia cells (AML), but the expression varies between patients and is highest for patients with a favorable prognosis after intensive chemotherapy. We therefore investigated the functional effects of two V-ATPase inhibitors (bafilomycin A1, concanamycin A) for primary AML cells derived from 80 consecutive patients. The V-ATPase inhibitors showed dose-dependent antiproliferative and proapoptotic effects that varied considerably between patients. A proteomic comparison of primary AML cells showing weak versus strong antiproliferative effects of V-ATPase inhibition showed a differential expression of proteins involved in intracellular transport/cytoskeleton functions, and an equivalent phosphoproteomic comparison showed a differential expression of proteins that regulate RNA processing/function together with increased activity of casein kinase 2. Patients with secondary AML, i.e., a heterogeneous subset with generally adverse prognosis and previous cytotoxic therapy, myeloproliferative neoplasia or myelodysplastic syndrome, were characterized by a strong antiproliferative effect of V-ATPase inhibition and also by a specific mRNA expression profile of V-ATPase interactome proteins. Furthermore, the V-ATPase inhibition altered the constitutive extracellular release of several soluble mediators (e.g., chemokines, interleukins, proteases, protease inhibitors), and increased mediator levels in the presence of AML-supporting bone marrow mesenchymal stem cells was then observed, especially for patients with secondary AML. Finally, animal studies suggested that the V-ATPase inhibitor bafilomycin had limited toxicity, even when combined with cytarabine. To conclude, V-ATPase inhibition has antileukemic effects in AML, but this effect varies between patients.Kreftforeningen, the Norwegian Cancer Society 100933Research Council of Norway 245979/F50Bergen Research FoundationNorSeq consortium, University of Berge

Functional analysis of ankrd55, a multiple sclerosis risk gene with unknown function.

213 p.El objetivo de este trabajo consiste en la caracterización de ANKRD55, un gen de función desconocida asociado a la esclerosis múltiple (EM). Para ello, por un lado, se realizó un análisis basado en DNA y expresión génica que consistió en la identificación de la principal fuente celular de ANKRD55 en PBMC, donde los resultados mostraron que los tres transcritos de ANKRD55 se expresaron de forma única y elevada en los linfocitos T CD4+. Además, el genotipo de riesgo para la variante intrónica asociada a EM mostró niveles significativamente más altos de dos transcritos de ANKRD55 en células T CD4+. A continuación, utilizando el sistema CRISPR/deadCas9 se analizaron 5 variantes de DNA presentes en ANKRD55 por su posible actividad enhancer, donde se observó que la región que contiene la variante rs71624119 regula la expresión de uno de los tres transcritos de ANKRD55. Por otro lado, mediante el análisis basado en proteína, se estudió la localización subcelular y la red de interacciones proteína-proteína de ANKRD55. Se observó que la localización intracelular de la forma endógena de ANKRD55 era principalmente nuclear en células inmunes y no inmunes, mientras que las formas recombinantes se encontraban tanto en el núcleo como en orgánulos membranosos y citosol de las líneas celulares HEK293 y HeLa. A través de la inmunoprecipitación (IP) de una de las formas recombinantes de ANKRD55 y análisis mediante espectrometría de masas, se identificaron 158 proteínas en el extracto proteico total y 22 en extracto nuclear que interaccionan con ANKRD55, de las cuales ocho se validaron por microscopía confocal e IP y posterior immunoblot