Neddylation regulates excitatory synaptic transmission and plasticity

Post-translational modifications, like phosphorylation, ubiquitylation, and sumoylation, have been shown to impact on synaptic neurotransmission by modifying pre- and postsynaptic proteins and therefore alter protein stability, localization, or protein-protein interactions. Previous studies showed that post-translational modifications are essential during the induction of synaptic plasticity, defined by a major reorganization of synaptic proteins. We demonstrated before that neddylation, a post-translational modification that covalently binds Nedd8 to lysine-residues, strongly affects neuronal maturation and spine stability. We now analysed the consequences of inhibiting neddylation on excitatory synaptic transmission and plasticity, which will help to narrow down possible targets, to make educated guesses, and test specific candidates. Here, we show that acute inhibition of neddylation impacts on synaptic neurotransmission before morphological changes occur. Our data indicate that pre- and postsynaptic proteins are neddylated since the inhibition of neddylation impacts on presynaptic release probability and postsynaptic receptor stabilization. In addition, blocking neddylation during the induction of long-term potentiation and long-term inhibition abolished both forms of synaptic plasticity. Therefore, this study shows the importance of identifying synaptic targets of the neddylation pathway to understand the regulation of synaptic transmission and plasticity.Fil: Brockmann, Marisa M.. Universitat Bonn; Alemania. Max Planck Institute Of Psychiatry; AlemaniaFil: Döngi, Michael. Universitat Bonn; AlemaniaFil: Einsfelder, Ulf. Universitat Bonn; AlemaniaFil: Körber, Nils. Universitat Bonn; AlemaniaFil: Refojo, Damian. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigación en Biomedicina de Buenos Aires - Instituto Partner de la Sociedad Max Planck; ArgentinaFil: Stein, Valentin. Universitat Bonn; Alemani

Cholinergic modulation of dentate gyrus processing through dynamic reconfiguration of inhibitory circuits

The dentate gyrus (DG) of the hippocampus plays a key role in memory formation, and it is known to be modulated by septal projections. By performing electrophysiology and optogenetics, we evaluated the role of cholinergic modulation in the processing of afferent inputs in the DG. We show that mature granule cells (GCs), but not adult-born immature neurons, have increased responses to afferent perforant path stimuli upon cholinergic modulation. This is due to a highly precise reconfiguration of inhibitory circuits, differentially affecting Parvalbumin and Somatostatin interneurons, resulting in a nicotinic-dependent perisomatic disinhibition of GCs. This circuit reorganization provides a mechanism by which mature GCs could escape the strong inhibition they receive, creating a window of opportunity for plasticity. Indeed, coincident activation of perforant path inputs with optogenetic release of acetylcholine produces a long-term potentiated response in GCs, essential for memory formation.Fil: Ogando, Mora. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigación en Biomedicina de Buenos Aires - Instituto Partner de la Sociedad Max Planck; ArgentinaFil: Pedroncini, Olivia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigación en Biomedicina de Buenos Aires - Instituto Partner de la Sociedad Max Planck; ArgentinaFil: Federman, Maria Noel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigación en Biomedicina de Buenos Aires - Instituto Partner de la Sociedad Max Planck; ArgentinaFil: Romano, Sebastián Alejo. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigación en Biomedicina de Buenos Aires - Instituto Partner de la Sociedad Max Planck; ArgentinaFil: Brum, Luciano Ariel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Lanuza, Guillermo Marcos. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Refojo, Damian. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigación en Biomedicina de Buenos Aires - Instituto Partner de la Sociedad Max Planck; ArgentinaFil: Marin Burgin, Antonia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigación en Biomedicina de Buenos Aires - Instituto Partner de la Sociedad Max Planck; Argentin

Neuroimmune and inflammatory signals in complex disorders of the central nervous system

An extensive microglial-astrocyte-monocyte-neuronal cross talk seems to be crucial for normal brain function, development, and recovery. However, under certain conditions neuroinflammatory interactions between brain cells and neuroimmune cells influence disease outcome and brain pathology. Microglial cells express a range of functional states with dynamically pleomorphic profiles from a surveilling status of synaptic transmission to an active player in major events of development such as synaptic elimination, regeneration, and repair. Also, inflammation mediates a series of neurotoxic roles in neuropsychiatric conditions and neurodegenerative diseases. The present review discusses data on the involvement of neuroinflammatory conditions that alter neuroimmune interactions in four different pathologies. In the first section of this review, we discuss the ability of the early developing brain to respond to a focal lesion with a rapid compensatory plasticity of intact axons and the role of microglial activation and proinflammatory cytokines in brain repair. In the second section, we present data of neuroinflammation and neurodegenerative disorders and discuss the role of reactive astrocytes in motor neuron toxicity and the progression of amyotrophic lateral sclerosis. In the third section, we discuss major depressive disorders as the consequence of dysfunctional interactions between neural and immune signals that result in increased peripheral immune responses and increase proinflammatory cytokines. In the last section, we discuss autism spectrum disorders and altered brain circuitries that emerge from abnormal long-term responses of innate inflammatory cytokines and microglial phenotypic dysfunctions.Fil: Liberman, Ana Clara. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigación en Biomedicina de Buenos Aires - Instituto Partner de la Sociedad Max Planck; ArgentinaFil: Trias, Emiliano. Instituto Pasteur de Montevideo; UruguayFil: Da Silva Chagas, Luana. Universidade Federal Fluminense; BrasilFil: Trindade, Pablo. No especifíca;Fil: Dos Santos Pereira, Marissol. Fundación Oswaldo Cruz; Brasil. Universidade Federal do Rio de Janeiro; BrasilFil: Refojo, Damian. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigación en Biomedicina de Buenos Aires - Instituto Partner de la Sociedad Max Planck; ArgentinaFil: Hedin Pereira, Cecilia. Universidade Federal do Rio de Janeiro; Brasil. Fundación Oswaldo Cruz; BrasilFil: Serfaty, Claudio A.. Universidade Federal Fluminense; Brasi

Co-Expression of Wild-Type P2X7R with Gln460Arg Variant Alters Receptor Function

The P2X7 receptor is a member of the P2X family of ligand-gated ion channels. A single-nucleotide polymorphism leading to a glutamine (Gln) by arginine (Arg) substitution at codon 460 of the purinergic P2X7 receptor (P2X7R) has been associated with mood disorders. No change in function (loss or gain) has been described for this SNP so far. Here we show that although the P2X7R-Gln460Arg variant per se is not compromised in its function, co-expression of wild-type P2X7R with P2X7R-Gln460Arg impairs receptor function with respect to calcium influx, channel currents and intracellular signaling in vitro. Moreover, co-immunoprecipitation and FRET studies show that the P2X7R-Gln460Arg variant physically interacts with P2X7R-WT. Specific silencing of either the normal or polymorphic variant rescues the heterozygous loss of function phenotype and restores normal function. The described loss of function due to co-expression, unique for mutations in the P2RX7 gene so far, explains the mechanism by which the P2X7R-Gln460Arg variant affects the normal function of the channel and may represent a mechanism of action for other mutations.Fil: Aprile García, Fernando. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Fisiología, Biología Molecular y Celular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigación en Biomedicina de Buenos Aires - Instituto Partner de la Sociedad Max Planck; ArgentinaFil: Metzger, Michael W.. Max Planck Institute of Psychiatry; AlemaniaFil: Paez Pereda, Marcelo. Max Planck Institute of Psychiatry; AlemaniaFil: Stadler, Herbert. Affectis Pharmaceuticals; AlemaniaFil: Acuña, Matias. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigación en Biomedicina de Buenos Aires - Instituto Partner de la Sociedad Max Planck; ArgentinaFil: Liberman, Ana Clara. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigación en Biomedicina de Buenos Aires - Instituto Partner de la Sociedad Max Planck; ArgentinaFil: Senin, Sergio Ariel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigación en Biomedicina de Buenos Aires - Instituto Partner de la Sociedad Max Planck; ArgentinaFil: Gerez, Juan Atilio. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigación en Biomedicina de Buenos Aires - Instituto Partner de la Sociedad Max Planck; ArgentinaFil: Hoijman, Esteban. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Centro de Microscopías Avanzadas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Refojo, Damian. Max Planck Institute of Psychiatry; Alemania. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Mitkovski, Mišo. Max Planck Institute of Experimental Medicine; AlemaniaFil: Panhuysen, Markus. Affectis Pharmaceuticals; AlemaniaFil: Stühmer, Walter. Max Planck Institute of Experimental Medicine; AlemaniaFil: Holsboer, Florian. Max Planck Institute of Psychiatry; Alemania. HMNC Brain Health; AlemaniaFil: Deussing, Jan M.. Max Planck Institute of Psychiatry; AlemaniaFil: Arzt, Eduardo Simon. Max Planck Institute of Psychiatry; Alemania. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigación en Biomedicina de Buenos Aires - Instituto Partner de la Sociedad Max Planck; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Fisiología, Biología Molecular y Celular; Argentin

B-Raf and CRHR1 internalization mediate biphasic ERK1/2 activation by CRH in hippocampal HT22 Cells.

CRH is a key regulator of neuroendocrine, autonomic, and behavioral response to stress. CRH-stimulated CRH receptor 1 (CRHR1) activates ERK1/2 depending on intracellular context. In a previous work, we demonstrated that CRH activates ERK1/2 in limbic areas of the mouse brain (hippocampus and basolateral amygdala). ERK1/2 is an essential mediator of hippocampal physiological processes including emotional behavior, synaptic plasticity, learning, and memory. To elucidate the molecular mechanisms by which CRH activates ERK1/2 in hippocampal neurons, we used the mouse hippocampal cell line HT22. We document for the first time that ERK1/2 activation in response to CRH is biphasic, involving a first cAMP- and B-Raf-dependent early phase and a second phase that critically depends on CRHR1 internalization and ß-arrestin2. By means of mass-spectrometry-based screening, we identified B-Raf-associated proteins that coimmunoprecipitate with endogenous B-Raf after CRHR1 activation. Using molecular and pharmacological tools, the functional impact of selected B-Raf partners in CRH-dependent ERK1/2 activation was dissected. These results indicate that 14-3-3 proteins, protein kinase A, and Rap1, are essential for early CRH-induced ERK1/2 activation, whereas dynamin and vimentin are required for the CRHR1 internalization-dependent phase. Both phases of ERK1/2 activation depend on calcium influx and are affected by calcium/calmodulin-dependent protein kinase II inactivation. Thus, this report describes the dynamics and biphasic nature of ERK1/2 activation downstream neuronal CRHR1 and identifies several new critical components of the CRHR1 signaling machinery that selectively controls the early and late phases of ERK1/2 activation, thus providing new potential therapeutic targets for stress-related disorders.Fil: Bonfiglio, Juan José. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque. Centenario. Instituto de Investigación En Biomedicina de Buenos Aires - Conicet -; Instituto P; Argentina;Fil: Inda, Carolina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque. Centenario. Instituto de Investigación En Biomedicina de Buenos Aires - Conicet -; Instituto P; Argentina;Fil: Senin, Sergio Ariel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque. Centenario. Instituto de Investigación En Biomedicina de Buenos Aires - Conicet -; Instituto P; Argentina;Fil: Maccarrone, Giuseppina. Max-Planck-Institut Für Psychiatrie; Alemania;Fil: Refojo, Damian. Max-Planck-Institut Für Psychiatrie; Alemania;Fil: Giacomini, Damiana Paula. Consejo Nacional de Invest.cientif.y Tecnicas. Oficina de Coordinacion Administrativa Pque. Centenario. Instituto de Invest.bioquimicas de Bs.as(i); Argentina;Fil: Turck, Christoph W.. Max-Planck-Institut Für Psychiatrie; Alemania;Fil: Holsboer, Florian. Max-Planck-Institut Für Psychiatrie; Alemania;Fil: Arzt, Eduardo Simon. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque. Centenario. Instituto de Investigación En Biomedicina de Buenos Aires - Conicet -; Instituto P; Argentina;Fil: Silberstein, Susana. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque. Centenario. Instituto de Investigación En Biomedicina de Buenos Aires - Conicet -; Instituto P; Argentina

Involvement of bone morphogenetic protein 4 (BMP-4) in pituitary prolactinoma pathogenesis through a Smad/estrogen receptor crosstalk

Pituitary tumor development involves clonal expansion stimulated by hormones and growth factors/cytokines. Using mRNA differential display, we found that the bone morphogenetic protein (BMP inhibitor noggin is down-regulated in prolactinomas from dopamine D2-receptor-deficient mice. BMP-4 is overexpressed in prolactinomas taken from dopamine D2-receptor-deficient female mice, but expression of the highly homologous BMP-2 does not differ in normal pituitary tissue and prolactinomas. BMP-4 is overexpressed in other prolactinoma models, including estradiol-induced rat prolactinomas and human prolactinomas, compared with normal tissue and other pituitary adenoma types (Western blot analysis of 48 tumors). BMP-4 stimulates, and noggin blocks, cell proliferation and the expression of c-Myc in human prolactinomas, whereas BMP-4 has no action in other human pituitary tumors. GH3 cells stably transfected with a dominant negative of Smad4 (Smad4dn; a BMP signal cotransducer) or noggin have reduced tumorigenicity in nude mice. Tumor growth recovered in vivo when the Smad4dn expression was lost, proving that BMP-4/Smad4 are involved in tumor development in vivo. BMP-4 and estrogens act through overlapping intracellular signaling mechanisms on GH3 cell proliferation and c-myc expression: they had additive effects at low concentrations but not at saturating doses, and their action was inhibited by blocking either pathway with the reciprocal antagonist (i.e., BMP-4 with ICI 182780 or 17β-estradiol with Smad4dn). Furthermore, coimmunoprecipitation studies demonstrate that under BMP-4 stimulation Smad4 and Smadl physically interact with the estrogen receptor. This previously undescribed prolactinoma pathogenesis mechanism may participate in tumorigenicity in other cells where estrogens and the type β transforming growth factor family have important roles.Facultad de Ciencias ExactasInstituto de Investigaciones Bioquímicas de La Plat

Chronic CRH depletion from GABAergic, long-range projection neurons in the extended amygdala reduces dopamine release and increases anxiety

The interplay between corticotropin-releasing hormone (CRH) and the dopaminergic system has predominantly been studied in addiction and reward, while CRH-dopamine interactions in anxiety are scarcely understood. We describe a new population of CRH-expressing, GABAergic, long-range-projecting neurons in the extended amygdala that innervate the ventral tegmental area and alter anxiety following chronic CRH depletion. These neurons are part of a distinct CRH circuit that acts anxiolytically by positively modulating dopamine release.Fil: Dedic, Nina. Max Planck Institute Of Psychiatry; AlemaniaFil: Kühne, Claudia. Max Planck Institute Of Psychiatry; AlemaniaFil: Jakovcevski, Mira. Max Planck Institute Of Psychiatry; AlemaniaFil: Hartmann, Jakob. Max Planck Institute Of Psychiatry; AlemaniaFil: Genewsky, Andreas J.. Max Planck Institut Of Psychiatry; AlemaniaFil: Gomes, Karina S.. Max Planck Institute Of Psychiatry; AlemaniaFil: Anderzhanova, Elmira. Max Planck Institute Of Psychiatry; AlemaniaFil: Pöhlmann, Max L.. Max Planck Institute Of Psychiatry; AlemaniaFil: Chang, Simon. Max Planck Institute Of Psychiatry; AlemaniaFil: Kolarz, Adam. Max Planck Institute Of Psychiatry; AlemaniaFil: Vogl, Annette M.. Max Planck Institute Of Psychiatry; AlemaniaFil: Dine, Julien. Max Planck Institute Of Psychiatry; AlemaniaFil: Metzger, Michael W.. Max Planck Institute of Psychiatry; ArmeniaFil: Schmid, Bianca. Max Planck Institute Of Psychiatry; AlemaniaFil: Almada, Rafael C.. Max Planck Institute Of Psychiatry; AlemaniaFil: Ressler, Kerry J.. Harvard Medical School; Estados UnidosFil: Wotjak, Carsten T.. Max Planck Institute Of Psychiatry; AlemaniaFil: Grinevich, Valery. University of Heidelberg; AlemaniaFil: Chen, Alon. Max Planck Institute Of Psychiatry; AlemaniaFil: Schmidt, Mathias V.. Institute Of Developmental Genetics, Helmholtz Zentrum; AlemaniaFil: Wurst, Wolfgang. German Center for Neurodegenerative Diseases; AlemaniaFil: Refojo, Damian. Max Planck Institute Of Psychiatry; Alemania. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigación en Biomedicina de Buenos Aires - Instituto Partner de la Sociedad Max Planck; ArgentinaFil: Deussing, Jan M.. Max Planck Institute Of Psychiatry; Alemani

Bi-allelic variants in NAE1 cause intellectual disability, ischiopubic hypoplasia, stress-mediated lymphopenia and neurodegeneration

Neddylation has been implicated in various cellular pathways and in the pathophysiology of numerous diseases. We identified four individuals with bi-allelic variants in NAE1, which encodes the neddylation E1 enzyme. Pathogenicity was supported by decreased NAE1 abundance and overlapping clinical and cellular phenotypes. To delineate how cellular consequences of NAE1 deficiency would lead to the clinical phenotype, we focused primarily on the rarest phenotypic features, based on the assumption that these would best reflect the pathophysiology at stake. Two of the rarest features, neuronal loss and lymphopenia worsening during infections, suggest that NAE1 is required during cellular stress caused by infections to protect against cell death. In support, we found that stressing the proteasome system with MG132—requiring upregulation of neddylation to restore proteasomal function and proteasomal stress—led to increased cell death in fibroblasts of individuals with NAE1 genetic variants. Additionally, we found decreased lymphocyte counts after CD3/CD28 stimulation and decreased NF-κB translocation in individuals with NAE1 variants. The rarest phenotypic feature—delayed closure of the ischiopubic rami—correlated with significant downregulation of RUN2X and SOX9 expression in transcriptomic data of fibroblasts. Both genes are involved in the pathophysiology of ischiopubic hypoplasia. Thus, we show that NAE1 plays a major role in (skeletal) development and cellular homeostasis during stress. Our approach suggests that a focus on rare phenotypic features is able to provide significant pathophysiological insights in diseases caused by mutations in genes with pleiotropic effects.</p

Bi-allelic variants in NAE1 cause intellectual disability, ischiopubic hypoplasia, stress-mediated lymphopenia and neurodegeneration

Neddylation has been implicated in various cellular pathways and in the pathophysiology of numerous diseases. We identified four individuals with bi-allelic variants in NAE1, which encodes the neddylation E1 enzyme. Pathogenicity was supported by decreased NAE1 abundance and overlapping clinical and cellular phenotypes. To delineate how cellular consequences of NAE1 deficiency would lead to the clinical phenotype, we focused primarily on the rarest phenotypic features, based on the assumption that these would best reflect the pathophysiology at stake. Two of the rarest features, neuronal loss and lymphopenia worsening during infections, suggest that NAE1 is required during cellular stress caused by infections to protect against cell death. In support, we found that stressing the proteasome system with MG132—requiring upregulation of neddylation to restore proteasomal function and proteasomal stress—led to increased cell death in fibroblasts of individuals with NAE1 genetic variants. Additionally, we found decreased lymphocyte counts after CD3/CD28 stimulation and decreased NF-κB translocation in individuals with NAE1 variants. The rarest phenotypic feature—delayed closure of the ischiopubic rami—correlated with significant downregulation of RUN2X and SOX9 expression in transcriptomic data of fibroblasts. Both genes are involved in the pathophysiology of ischiopubic hypoplasia. Thus, we show that NAE1 plays a major role in (skeletal) development and cellular homeostasis during stress. Our approach suggests that a focus on rare phenotypic features is able to provide significant pathophysiological insights in diseases caused by mutations in genes with pleiotropic effects.</p

Bi-allelic variants in NAE1 cause intellectual disability, ischiopubic hypoplasia, stress-mediated lymphopenia and neurodegeneration

Neddylation has been implicated in various cellular pathways and in the pathophysiology of numerous diseases. We identified four individuals with bi-allelic variants in NAE1, which encodes the neddylation E1 enzyme. Pathogenicity was supported by decreased NAE1 abundance and overlapping clinical and cellular phenotypes. To delineate how cellular consequences of NAE1 deficiency would lead to the clinical phenotype, we focused primarily on the rarest phenotypic features, based on the assumption that these would best reflect the pathophysiology at stake. Two of the rarest features, neuronal loss and lymphopenia worsening during infections, suggest that NAE1 is required during cellular stress caused by infections to protect against cell death. In support, we found that stressing the proteasome system with MG132—requiring upregulation of neddylation to restore proteasomal function and proteasomal stress—led to increased cell death in fibroblasts of individuals with NAE1 genetic variants. Additionally, we found decreased lymphocyte counts after CD3/CD28 stimulation and decreased NF-κB translocation in individuals with NAE1 variants. The rarest phenotypic feature—delayed closure of the ischiopubic rami—correlated with significant downregulation of RUN2X and SOX9 expression in transcriptomic data of fibroblasts. Both genes are involved in the pathophysiology of ischiopubic hypoplasia. Thus, we show that NAE1 plays a major role in (skeletal) development and cellular homeostasis during stress. Our approach suggests that a focus on rare phenotypic features is able to provide significant pathophysiological insights in diseases caused by mutations in genes with pleiotropic effects.</p