66 results on '"Margherita Mutarelli"'
Search Results
2. EGR1 drives cell proliferation by directly stimulating TFEB transcription in response to starvation.
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Marcella Cesana, Gennaro Tufano, Francesco Panariello, Nicolina Zampelli, Susanna Ambrosio, Rossella De Cegli, Margherita Mutarelli, Lorenzo Vaccaro, Micheal J Ziller, Davide Cacchiarelli, Diego L Medina, and Andrea Ballabio
- Subjects
Biology (General) ,QH301-705.5 - Abstract
The stress-responsive transcription factor EB (TFEB) is a master controller of lysosomal biogenesis and autophagy and plays a major role in several cancer-associated diseases. TFEB is regulated at the posttranslational level by the nutrient-sensitive kinase complex mTORC1. However, little is known about the regulation of TFEB transcription. Here, through integrative genomic approaches, we identify the immediate-early gene EGR1 as a positive transcriptional regulator of TFEB expression in human cells and demonstrate that, in the absence of EGR1, TFEB-mediated transcriptional response to starvation is impaired. Remarkably, both genetic and pharmacological inhibition of EGR1, using the MEK1/2 inhibitor Trametinib, significantly reduced the proliferation of 2D and 3D cultures of cells displaying constitutive activation of TFEB, including those from a patient with Birt-Hogg-Dubé (BHD) syndrome, a TFEB-driven inherited cancer condition. Overall, we uncover an additional layer of TFEB regulation consisting in modulating its transcription via EGR1 and propose that interfering with the EGR1-TFEB axis may represent a therapeutic strategy to counteract constitutive TFEB activation in cancer-associated conditions.
- Published
- 2023
- Full Text
- View/download PDF
3. TFEB regulates murine liver cell fate during development and regeneration
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Nunzia Pastore, Tuong Huynh, Niculin J. Herz, Alessia Calcagni’, Tiemo J. Klisch, Lorenzo Brunetti, Kangho Ho Kim, Marco De Giorgi, Ayrea Hurley, Annamaria Carissimo, Margherita Mutarelli, Niya Aleksieva, Luca D’Orsi, William R. Lagor, David D. Moore, Carmine Settembre, Milton J. Finegold, Stuart J. Forbes, and Andrea Ballabio
- Subjects
Science - Abstract
The Transcription Factor EB (TFEB) is known to regulate cellular homeostasis and energy metabolism, but its role in cell fate determination in the liver is unknown. Here, the authors show that TFEB regulates the progenitor/cholangiocyte lineage and that its depletion prevents tissue recovery upon injury.
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- 2020
- Full Text
- View/download PDF
4. Myopalladin promotes muscle growth through modulation of the serum response factor pathway
- Author
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Maria Carmela Filomena, Daniel L. Yamamoto, Marco Caremani, Vinay K. Kadarla, Giuseppina Mastrototaro, Simone Serio, Anupama Vydyanath, Margherita Mutarelli, Arcamaria Garofalo, Irene Pertici, Ralph Knöll, Vincenzo Nigro, Pradeep K. Luther, Richard L. Lieber, Moriah R. Beck, Marco Linari, and Marie‐Louise Bang
- Subjects
Skeletal muscle ,Sarcomere ,Knockout mouse ,Muscle growth ,Actin dynamics ,Serum response factor pathway ,Diseases of the musculoskeletal system ,RC925-935 ,Human anatomy ,QM1-695 - Abstract
Abstract Background Myopalladin (MYPN) is a striated muscle‐specific, immunoglobulin‐containing protein located in the Z‐line and I‐band of the sarcomere as well as the nucleus. Heterozygous MYPN gene mutations are associated with hypertrophic, dilated, and restrictive cardiomyopathy, and homozygous loss‐of‐function truncating mutations have recently been identified in patients with cap myopathy, nemaline myopathy, and congenital myopathy with hanging big toe. Methods Constitutive MYPN knockout (MKO) mice were generated, and the role of MYPN in skeletal muscle was studied through molecular, cellular, biochemical, structural, biomechanical, and physiological studies in vivo and in vitro. Results MKO mice were 13% smaller compared with wild‐type controls and exhibited a 48% reduction in myofibre cross‐sectional area (CSA) and significantly increased fibre number. Similarly, reduced myotube width was observed in MKO primary myoblast cultures. Biomechanical studies showed reduced isometric force and power output in MKO mice as a result of the reduced CSA, whereas the force developed by each myosin molecular motor was unaffected. While the performance by treadmill running was similar in MKO and wild‐type mice, MKO mice showed progressively decreased exercise capability, Z‐line damage, and signs of muscle regeneration following consecutive days of downhill running. Additionally, MKO muscle exhibited progressive Z‐line widening starting from 8 months of age. RNA‐sequencing analysis revealed down‐regulation of serum response factor (SRF)‐target genes in muscles from postnatal MKO mice, important for muscle growth and differentiation. The SRF pathway is regulated by actin dynamics as binding of globular actin to the SRF‐cofactor myocardin‐related transcription factor A (MRTF‐A) prevents its translocation to the nucleus where it binds and activates SRF. MYPN was found to bind and bundle filamentous actin as well as interact with MRTF‐A. In particular, while MYPN reduced actin polymerization, it strongly inhibited actin depolymerization and consequently increased MRTF‐A‐mediated activation of SRF signalling in myogenic cells. Reduced myotube width in MKO primary myoblast cultures was rescued by transduction with constitutive active SRF, demonstrating that MYPN promotes skeletal muscle growth through activation of the SRF pathway. Conclusions Myopalladin plays a critical role in the control of skeletal muscle growth through its effect on actin dynamics and consequently the SRF pathway. In addition, MYPN is important for the maintenance of Z‐line integrity during exercise and aging. These results suggest that muscle weakness in patients with biallelic MYPN mutations may be associated with reduced myofibre CSA and SRF signalling and that the disease phenotype may be aggravated by exercise.
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- 2020
- Full Text
- View/download PDF
5. The Aquatic Invertebrate Hydra vulgaris Releases Molecular Messages Through Extracellular Vesicles
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Maria Moros, Eugenio Fergola, Valentina Marchesano, Margherita Mutarelli, Giuseppina Tommasini, Beata Miedziak, Giuliana Palumbo, Alfredo Ambrosone, Angela Tino, and Claudia Tortiglione
- Subjects
extracellular vesicles ,cell-cell communication ,exosome ,RNA-seq ,Hydra vulgaris ,aquatic invertebrate model ,Biology (General) ,QH301-705.5 - Abstract
Recent body of evidence demonstrates that extracellular vesicles (EVs) represent the first language of cell-cell communication emerged during evolution. In aquatic environments, transferring signals between cells by EVs offers protection against degradation, allowing delivering of chemical information in high local concentrations to the target cells. The packaging of multiple signals, including those of hydrophobic nature, ensures target cells to receive the same EV-conveyed messages, and the coordination of a variety of physiological processes across cells of a single organisms, or at the population level, i.e., mediating the population’s response to changing environmental conditions. Here, we purified EVs from the medium of the freshwater invertebrate Hydra vulgaris, and the molecular profiling by proteomic and transcriptomic analyses revealed multiple markers of the exosome EV subtype, from structural proteins to stress induced messages promoting cell survival. Moreover, positive and negative regulators of the Wnt/β-catenin signaling pathway, the major developmental pathway acting in body axial patterning, were identified. Functional analysis on amputated polyps revealed EV ability to modulate both head and foot regeneration, suggesting bioactivity of the EV cargo and opening new perspectives on the mechanisms of developmental signalling. Our results open the path to unravel EV biogenesis and function in all cnidarian species, tracing back the origin of the cell-cell, cross-species or cross-kingdom communication in aquatic ecosystems.
- Published
- 2021
- Full Text
- View/download PDF
6. Effect of CB2 Stimulation on Gene Expression in Pediatric B-Acute Lymphoblastic Leukemia: New Possible Targets
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Francesca Punzo, Maura Argenziano, Chiara Tortora, Alessandra Di Paola, Margherita Mutarelli, Elvira Pota, Martina Di Martino, Daniela Di Pinto, Maria Maddalena Marrapodi, Domenico Roberti, and Francesca Rossi
- Subjects
acute lymphoblastic leukemia ,SUP-B15 cell line ,endocannabinoid system ,RNA sequencing ,CB2 receptors ,Biology (General) ,QH301-705.5 ,Chemistry ,QD1-999 - Abstract
Acute lymphoblastic leukemia type B (B-ALL) is the most common kind of pediatric leukemia, characterized by the clonal proliferation of type B lymphoid stem cells. Important progress in ALL treatments led to improvements in long-term survival; nevertheless, many adverse long-term consequences still concern the medical community. Molecular and cellular target therapies, together with immunotherapy, are promising strategies to overcome these concerns. Cannabinoids, enzymes involved in their metabolism, and cannabinoid receptors type 1 (CB1) and type 2 (CB2) constitute the endocannabinoid system, involved in inflammation, immune response, and cancer. CB2 receptor stimulation exerts anti-proliferative and anti-invasive effects in many tumors. In this study, we evaluated the effects of CB2 stimulation on B-ALL cell lines, SUP-B15, by RNA sequencing, Western blotting, and ELISA. We observe a lower expression of CB2 in SUP-B15 cells compared to lymphocytes from healthy subjects, hypothesizing its involvement in B-ALL pathogenesis. CB2 stimulation reduces the expression of CD9, SEC61G, TBX21, and TMSB4X genes involved in tumor growth and progression, and also negatively affects downstream intracellular pathways. Our findings suggest an antitumor role of CB2 stimulation in B-ALL, and highlight a functional correlation between CB2 receptors and specific anti-tumoral pathways, even though further investigations are needed.
- Published
- 2022
- Full Text
- View/download PDF
7. Comprehensive kinome NGS targeted expression profiling by KING-REX
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Giovanni Carapezza, Carlo Cusi, Ettore Rizzo, Laura Raddrizzani, Sebastiano Di Bella, Alessio Somaschini, Antonella Leone, Rosita Lupi, Margherita Mutarelli, Vincenzo Nigro, Diego di Bernardo, Paolo Magni, Antonella Isacchi, and Roberta Bosotti
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Kinome gene expression ,NGS RNA targeted panel ,Biotechnology ,TP248.13-248.65 ,Genetics ,QH426-470 - Abstract
Abstract Background Protein kinases are enzymes controlling different cellular functions. Genetic alterations often result in kinase dysregulation, making kinases a very attractive class of druggable targets in several human diseases. Existing approved drugs still target a very limited portion of the human ‘kinome’, demanding a broader functional knowledge of individual and co-expressed kinase patterns in physiologic and pathologic settings. The development of novel rapid and cost-effective methods for kinome screening is therefore highly desirable, potentially leading to the identification of novel kinase drug targets. Results In this work, we describe the development of KING-REX (KINase Gene RNA EXpression), a comprehensive kinome RNA targeted custom assay-based panel designed for Next Generation Sequencing analysis, coupled with a dedicated data analysis pipeline. We have conceived KING-REX for the gene expression analysis of 512 human kinases; for 319 kinases, paired assays and custom analysis pipeline features allow the evaluation of 3′- and 5′-end transcript imbalances as readout for the prediction of gene rearrangements. Validation tests on cell line models harboring known gene fusions demonstrated a comparable accuracy of KING-REX gene expression assessment as in whole transcriptome analyses, together with a robust detection of transcript portion imbalances in rearranged kinases, even in complex RNA mixtures or in degraded RNA. Conclusions These results support the use of KING-REX as a rapid and cost effective kinome investigation tool in the field of kinase target identification for applications in cancer biology and other human diseases.
- Published
- 2019
- Full Text
- View/download PDF
8. Next-generation sequencing identifies transportin 3 as the causative gene for LGMD1F.
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Annalaura Torella, Marina Fanin, Margherita Mutarelli, Enrico Peterle, Francesca Del Vecchio Blanco, Rossella Rispoli, Marco Savarese, Arcomaria Garofalo, Giulio Piluso, Lucia Morandi, Giulia Ricci, Gabriele Siciliano, Corrado Angelini, and Vincenzo Nigro
- Subjects
Medicine ,Science - Abstract
Limb-girdle muscular dystrophies (LGMD) are genetically and clinically heterogeneous conditions. We investigated a large family with autosomal dominant transmission pattern, previously classified as LGMD1F and mapped to chromosome 7q32. Affected members are characterized by muscle weakness affecting earlier the pelvic girdle and the ileopsoas muscles. We sequenced the whole exome of four family members and identified a shared heterozygous frame-shift variant in the Transportin 3 (TNPO3) gene, encoding a member of the importin-β super-family. The TNPO3 gene is mapped within the LGMD1F critical interval and its 923-amino acid human gene product is also expressed in skeletal muscle. In addition, we identified an isolated case of LGMD with a new missense mutation in the same gene. We localized the mutant TNPO3 around the nucleus, but not inside. The involvement of gene related to the nuclear transport suggests a novel disease mechanism leading to muscular dystrophy.
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- 2013
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9. Molecular diagnosis of Usher syndrome: application of two different next generation sequencing-based procedures.
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Danilo Licastro, Margherita Mutarelli, Ivana Peluso, Kornelia Neveling, Nienke Wieskamp, Rossella Rispoli, Diego Vozzi, Emmanouil Athanasakis, Angela D'Eustacchio, Mariateresa Pizzo, Francesca D'Amico, Carmela Ziviello, Francesca Simonelli, Antonella Fabretto, Hans Scheffer, Paolo Gasparini, Sandro Banfi, and Vincenzo Nigro
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Medicine ,Science - Abstract
Usher syndrome (USH) is a clinically and genetically heterogeneous disorder characterized by visual and hearing impairments. Clinically, it is subdivided into three subclasses with nine genes identified so far. In the present study, we investigated whether the currently available Next Generation Sequencing (NGS) technologies are already suitable for molecular diagnostics of USH. We analyzed a total of 12 patients, most of which were negative for previously described mutations in known USH genes upon primer extension-based microarray genotyping. We enriched the NGS template either by whole exome capture or by Long-PCR of the known USH genes. The main NGS sequencing platforms were used: SOLiD for whole exome sequencing, Illumina (Genome Analyzer II) and Roche 454 (GS FLX) for the Long-PCR sequencing. Long-PCR targeting was more efficient with up to 94% of USH gene regions displaying an overall coverage higher than 25×, whereas whole exome sequencing yielded a similar coverage for only 50% of those regions. Overall this integrated analysis led to the identification of 11 novel sequence variations in USH genes (2 homozygous and 9 heterozygous) out of 18 detected. However, at least two cases were not genetically solved. Our result highlights the current limitations in the diagnostic use of NGS for USH patients. The limit for whole exome sequencing is linked to the need of a strong coverage and to the correct interpretation of sequence variations with a non obvious, pathogenic role, whereas the targeted approach suffers from the high genetic heterogeneity of USH that may be also caused by the presence of additional causative genes yet to be identified.
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- 2012
- Full Text
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10. Massive-scale RNA-Seq analysis of non ribosomal transcriptome in human trisomy 21.
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Valerio Costa, Claudia Angelini, Luciana D'Apice, Margherita Mutarelli, Amelia Casamassimi, Linda Sommese, Maria Assunta Gallo, Marianna Aprile, Roberta Esposito, Luigi Leone, Aldo Donizetti, Stefania Crispi, Monica Rienzo, Berardo Sarubbi, Raffaele Calabrò, Marco Picardi, Paola Salvatore, Teresa Infante, Piergiuseppe De Berardinis, Claudio Napoli, and Alfredo Ciccodicola
- Subjects
Medicine ,Science - Abstract
Hybridization- and tag-based technologies have been successfully used in Down syndrome to identify genes involved in various aspects of the pathogenesis. However, these technologies suffer from several limits and drawbacks and, to date, information about rare, even though relevant, RNA species such as long and small non-coding RNAs, is completely missing. Indeed, none of published works has still described the whole transcriptional landscape of Down syndrome. Although the recent advances in high-throughput RNA sequencing have revealed the complexity of transcriptomes, most of them rely on polyA enrichment protocols, able to detect only a small fraction of total RNA content. On the opposite end, massive-scale RNA sequencing on rRNA-depleted samples allows the survey of the complete set of coding and non-coding RNA species, now emerging as novel contributors to pathogenic mechanisms. Hence, in this work we analysed for the first time the complete transcriptome of human trisomic endothelial progenitor cells to an unprecedented level of resolution and sensitivity by RNA-sequencing. Our analysis allowed us to detect differential expression of even low expressed genes crucial for the pathogenesis, to disclose novel regions of active transcription outside yet annotated loci, and to investigate a plethora of non-polyadenylated long as well as short non coding RNAs. Novel splice isoforms for a large subset of crucial genes, and novel extended untranslated regions for known genes--possibly novel miRNA targets or regulatory sites for gene transcription--were also identified in this study. Coupling the rRNA depletion of samples, followed by high-throughput RNA-sequencing, to the easy availability of these cells renders this approach very feasible for transcriptome studies, offering the possibility of investigating in-depth blood-related pathological features of Down syndrome, as well as other genetic disorders.
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- 2011
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11. VarGenius executes cohort-level DNA-seq variant calling and annotation and allows to manage the resulting data through a PostgreSQL database.
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Francesco Musacchia, Andrea Ciolfi, Margherita Mutarelli, Alessandro Bruselles, Raffaele Castello, Michele Pinelli, Swaraj Basu, Sandro Banfi, Giorgio Casari, Marco Tartaglia, and Vincenzo Nigro
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- 2018
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12. Esrrb conveys naïve pluripotent cells through the formative transcriptional program
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Elena Carbognin, Valentina Carlini, Francesco Panariello, Martina Chieregato, Elena Guerzoni, Davide Benvegnù, Valentina Perrera, Cristina Malucelli, Marcella Cesana, Antonio Grimaldi, Margherita Mutarelli, Annamaria Carissimo, Eitan Tennenbaum, Hillel Kugler, Jamie A. Hackett, Davide Cacchiarelli, Graziano Martello, Carbognin, Elena, Carlini, Valentina, Panariello, Francesco, Chieregato, Martina, Guerzoni, Elena, Benvegnù, Davide, Perrera, Valentina, Malucelli, Cristina, Cesana, Marcella, Grimaldi, Antonio, Mutarelli, Margherita, Carissimo, Annamaria, Tennenbaum, Eitan, Kugler, Hillel, Hackett, Jamie A., Cacchiarelli, Davide, and Martello, Graziano
- Abstract
Pluripotency is the potential of a single cell to give rise to all embryonic lineages and first emerges in the naïve epiblast of the preimplantation embryo. Upon implantation, epiblast cells transit to a formative phase, which is preparatory for their differentiation into all somatic lineages and primordial germ cells (PGCs). Murine naïve embryonic stem cells (ESCs) recapitulate the molecular and functional properties of the naïve epiblast, including the capacity to acquire a formative state and differentiate. However, the transition to the formative state, and its functional relevance, is still heavily investigated. Here we observed that Esrrb, a pivotal naïve pluripotency factor, is both required and sufficient to activate formative genes. In naïve cells ESRRB occupies both naïve and formative gene loci. During the formative transition, however, ESRRB binding becomes consolidated on formative genes. Subsequently, ESRRB occupancy is mostly lost, and the formative transcriptional program is inactivated. Functionally, genetic inactivation of Esrrb leads to impaired PGC specification, spontaneous expression of mesendoderm and trophectoderm markers, and inability to generate Formative Stem (FS) cells. Moreover, the 3D organisation in a polarised epithelium with a central lumen, as observed in the formative epiblast, is impaired in the absence of Esrrb. Thus, Esrrb is critical for activating the formative transition and consequently for executing timely and unbiased multilineage differentiation and self-organisation of murine pluripotent cells.
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- 2023
13. Esrrb guides naive pluripotent cells through the formative transcriptional programme
- Author
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Elena Carbognin, Valentina Carlini, Francesco Panariello, Martina Chieregato, Elena Guerzoni, Davide Benvegnù, Valentina Perrera, Cristina Malucelli, Marcella Cesana, Antonio Grimaldi, Margherita Mutarelli, Annamaria Carissimo, Eitan Tannenbaum, Hillel Kugler, Jamie A. Hackett, Davide Cacchiarelli, and Graziano Martello
- Subjects
Formative pluripotency ,pluripotency, Formative pluripotency, Esrrb, PGC specification, Cell fate, Gene regulatory network ,Cell fate ,Gene regulatory network ,Cell Biology ,Esrrb ,pluripotency ,PGC specification - Published
- 2023
14. Myopalladin promotes muscle growth through modulation of the serum response factor pathway
- Author
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Marie Louise Bang, Daniel L. Yamamoto, Giuseppina Mastrototaro, Simone Serio, Marco Caremani, Richard L. Lieber, Ralph Knöll, Anupama Vydyanath, Marco Linari, Arcamaria Garofalo, Vinay Kumar Kadarla, Vincenzo Nigro, Irene Pertici, Maria Carmela Filomena, Margherita Mutarelli, Moriah R. Beck, Pradeep K. Luther, Filomena, Maria Carmela, Yamamoto, Daniel L, Caremani, Marco, Kadarla, Vinay K, Mastrototaro, Giuseppina, Serio, Simone, Vydyanath, Anupama, Mutarelli, Margherita, Garofalo, Arcamaria, Pertici, Irene, Knöll, Ralph, Nigro, Vincenzo, Luther, Pradeep K, Lieber, Richard L, Beck, Moriah R, Linari, Marco, and Bang, Marie-Louise
- Subjects
0301 basic medicine ,Serum Response Factor ,lcsh:Diseases of the musculoskeletal system ,Actin dynamics ,Knockout mouse ,Muscle growth ,Sarcomere ,Serum response factor pathway ,Skeletal muscle ,Muscle Proteins ,Filamentous actin ,lcsh:QM1-695 ,Mice ,03 medical and health sciences ,0302 clinical medicine ,Physiology (medical) ,Serum response factor ,Myosin ,Animals ,Humans ,Medicine ,Myocyte ,Orthopedics and Sports Medicine ,Muscle, Skeletal ,Actin ,Mice, Knockout ,Actin dynamic ,business.industry ,Original Articles ,MYPN ,lcsh:Human anatomy ,Cell biology ,030104 developmental biology ,medicine.anatomical_structure ,030220 oncology & carcinogenesis ,Female ,Original Article ,lcsh:RC925-935 ,business - Abstract
Background Myopalladin (MYPN) is a striated muscle‐specific, immunoglobulin‐containing protein located in the Z‐line and I‐band of the sarcomere as well as the nucleus. Heterozygous MYPN gene mutations are associated with hypertrophic, dilated, and restrictive cardiomyopathy, and homozygous loss‐of‐function truncating mutations have recently been identified in patients with cap myopathy, nemaline myopathy, and congenital myopathy with hanging big toe. Methods Constitutive MYPN knockout (MKO) mice were generated, and the role of MYPN in skeletal muscle was studied through molecular, cellular, biochemical, structural, biomechanical, and physiological studies in vivo and in vitro. Results MKO mice were 13% smaller compared with wild‐type controls and exhibited a 48% reduction in myofibre cross‐sectional area (CSA) and significantly increased fibre number. Similarly, reduced myotube width was observed in MKO primary myoblast cultures. Biomechanical studies showed reduced isometric force and power output in MKO mice as a result of the reduced CSA, whereas the force developed by each myosin molecular motor was unaffected. While the performance by treadmill running was similar in MKO and wild‐type mice, MKO mice showed progressively decreased exercise capability, Z‐line damage, and signs of muscle regeneration following consecutive days of downhill running. Additionally, MKO muscle exhibited progressive Z‐line widening starting from 8 months of age. RNA‐sequencing analysis revealed down‐regulation of serum response factor (SRF)‐target genes in muscles from postnatal MKO mice, important for muscle growth and differentiation. The SRF pathway is regulated by actin dynamics as binding of globular actin to the SRF‐cofactor myocardin‐related transcription factor A (MRTF‐A) prevents its translocation to the nucleus where it binds and activates SRF. MYPN was found to bind and bundle filamentous actin as well as interact with MRTF‐A. In particular, while MYPN reduced actin polymerization, it strongly inhibited actin depolymerization and consequently increased MRTF‐A‐mediated activation of SRF signalling in myogenic cells. Reduced myotube width in MKO primary myoblast cultures was rescued by transduction with constitutive active SRF, demonstrating that MYPN promotes skeletal muscle growth through activation of the SRF pathway. Conclusions Myopalladin plays a critical role in the control of skeletal muscle growth through its effect on actin dynamics and consequently the SRF pathway. In addition, MYPN is important for the maintenance of Z‐line integrity during exercise and aging. These results suggest that muscle weakness in patients with biallelic MYPN mutations may be associated with reduced myofibre CSA and SRF signalling and that the disease phenotype may be aggravated by exercise.
- Published
- 2020
15. The Aquatic Invertebrate
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Maria, Moros, Eugenio, Fergola, Valentina, Marchesano, Margherita, Mutarelli, Giuseppina, Tommasini, Beata, Miedziak, Giuliana, Palumbo, Alfredo, Ambrosone, Angela, Tino, and Claudia, Tortiglione
- Subjects
Cell and Developmental Biology ,exosome ,aquatic invertebrate model ,RNA-seq ,Hydra vulgaris ,extracellular vesicles ,cell-cell communication ,Original Research - Abstract
Recent body of evidence demonstrates that extracellular vesicles (EVs) represent the first language of cell-cell communication emerged during evolution. In aquatic environments, transferring signals between cells by EVs offers protection against degradation, allowing delivering of chemical information in high local concentrations to the target cells. The packaging of multiple signals, including those of hydrophobic nature, ensures target cells to receive the same EV-conveyed messages, and the coordination of a variety of physiological processes across cells of a single organisms, or at the population level, i.e., mediating the population’s response to changing environmental conditions. Here, we purified EVs from the medium of the freshwater invertebrate Hydra vulgaris, and the molecular profiling by proteomic and transcriptomic analyses revealed multiple markers of the exosome EV subtype, from structural proteins to stress induced messages promoting cell survival. Moreover, positive and negative regulators of the Wnt/β-catenin signaling pathway, the major developmental pathway acting in body axial patterning, were identified. Functional analysis on amputated polyps revealed EV ability to modulate both head and foot regeneration, suggesting bioactivity of the EV cargo and opening new perspectives on the mechanisms of developmental signalling. Our results open the path to unravel EV biogenesis and function in all cnidarian species, tracing back the origin of the cell-cell, cross-species or cross-kingdom communication in aquatic ecosystems.
- Published
- 2021
16. Tracing back the origin of cell-cell communication: Hydra vulgaris releases extracellular vesicles delivering regulators of head and foot regeneration
- Author
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Giuseppina Tommasini, Eugenio Fergola, Giuliana Palumbo, Valentina Marchesano, Claudia Tortiglione, Angela Tino, María Moros, Beata Miedziak, Margherita Mutarelli, and Alfredo Ambrosone
- Subjects
Cell signaling ,medicine.anatomical_structure ,Regeneration (biology) ,Cell ,Hydra vulgaris ,medicine ,Head (vessel) ,Biology ,biology.organism_classification ,Extracellular vesicles ,Cell biology - Abstract
Recent body of evidence demonstrates that extracellular vesicles (EVs) represent the first language of cell-cell communication emerged during evolution. In aquatic environments, transferring signals between cells by EVs offer protection against degradation, allowing delivering of chemical information in high local concentrations to the target cells. The packaging of multiple signals, including those of hydrophobic nature, ensures target cells to receive the same EV-conveyed messages, and the coordination of a variety of physiological processes across cells of a single organisms, or at the population level, i.e. mediating the population´s response to changing environmental conditions. Here, we purified EVs from the medium of the freshwater invertebrate Hydra vulgaris, and the molecular profiling by proteomic and transcriptomic analyses revealed multiple markers of the exosome EV subtype. Moreover, positive and negative regulators of the Wnt/β-catenin signaling pathway, the major developmental pathway acting in body axial patterning, were identified. Functional analysis on amputated polyps revealed EV ability to interfere with both head and foot regeneration, suggesting an active role in setting up tissue gradients and oro-aboral polarity through delivery of short and long-distance signals. Our results open the path to unravel EV biogenesis and function in all cnidarian species, tracing back the origin of the cell-cell, cross-species or cross-kingdom communication in aquatic ecosystems
- Published
- 2021
17. Comprehensive kinome NGS targeted expression profiling by KING-REX
- Author
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Ettore Rizzo, Vincenzo Nigro, Alessio Somaschini, Sebastiano Di Bella, Antonella Leone, Margherita Mutarelli, Giovanni Carapezza, Diego di Bernardo, Laura Raddrizzani, Roberta Bosotti, Rosita Lupi, Antonella Isacchi, Carlo Cusi, Paolo Magni, Carapezza, G, Cusi, C, Rizzo, E, Raddrizzani, L, Di Bella, S, Somaschini, A, Leone, A, Lupi, R, Mutarelli, M, Nigro, V, di Bernardo, D, Magni, P, Isacchi, A, Bosotti, R, Carapezza, G., Cusi, C., Rizzo, E., Raddrizzani, L., Di Bella, S., Somaschini, A., Leone, A., Lupi, R., Mutarelli, M., Nigro, V., Di Bernardo, D., Magni, P., Isacchi, A., and Bosotti, R.
- Subjects
0106 biological sciences ,lcsh:QH426-470 ,RNA Stability ,lcsh:Biotechnology ,Kinome gene expression ,Druggability ,Computational biology ,Biology ,Proteomics ,01 natural sciences ,NGS RNA targeted panel ,Transcriptome ,03 medical and health sciences ,lcsh:TP248.13-248.65 ,Gene expression ,Genetics ,Kinome ,Gene ,030304 developmental biology ,0303 health sciences ,Methodology Article ,Gene Expression Profiling ,High-Throughput Nucleotide Sequencing ,Gene expression profiling ,lcsh:Genetics ,DNA microarray ,Gene Fusion ,Protein Kinases ,010606 plant biology & botany ,Biotechnology - Abstract
Background Protein kinases are enzymes controlling different cellular functions. Genetic alterations often result in kinase dysregulation, making kinases a very attractive class of druggable targets in several human diseases. Existing approved drugs still target a very limited portion of the human ‘kinome’, demanding a broader functional knowledge of individual and co-expressed kinase patterns in physiologic and pathologic settings. The development of novel rapid and cost-effective methods for kinome screening is therefore highly desirable, potentially leading to the identification of novel kinase drug targets. Results In this work, we describe the development of KING-REX (KINase Gene RNA EXpression), a comprehensive kinome RNA targeted custom assay-based panel designed for Next Generation Sequencing analysis, coupled with a dedicated data analysis pipeline. We have conceived KING-REX for the gene expression analysis of 512 human kinases; for 319 kinases, paired assays and custom analysis pipeline features allow the evaluation of 3′- and 5′-end transcript imbalances as readout for the prediction of gene rearrangements. Validation tests on cell line models harboring known gene fusions demonstrated a comparable accuracy of KING-REX gene expression assessment as in whole transcriptome analyses, together with a robust detection of transcript portion imbalances in rearranged kinases, even in complex RNA mixtures or in degraded RNA. Conclusions These results support the use of KING-REX as a rapid and cost effective kinome investigation tool in the field of kinase target identification for applications in cancer biology and other human diseases. Electronic supplementary material The online version of this article (10.1186/s12864-019-5676-3) contains supplementary material, which is available to authorized users.
- Published
- 2019
18. SB4ER: an ELIXIR Service Bundle for Epidemic Response
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CASTRENSE SAVOJARDO, Pier Luigi Martelli, Giulia Babbi, Marco Anteghini, Matteo Manfredi, Giovanni Madeo, Emidio Capriotti, Jumamurat R. Bayjanov, Margherita Mutarelli, and Rita Casadio
- Abstract
Epidemic spread of new pathogens is quite a frequent event that affects not only humans but also animals and plants, and specifically livestock and crops. In the last few years, many novel pathogenic viruses have threatened human life. Some were mutations of the traditional influenza viruses, and some were viruses that crossed the animal-human divide.In both cases, when a novel virus or bacterial strain for which there is no pre-existing immunity or a vaccine released, there is the possibility of an epidemic or even a pandemic event, as the one we are experiencing today with COVID-19.In this context, we defined an ELIXIR Service Bundle for Epidemic Response: a set of tools and workflows to facilitate and speed up the study of new pathogens, viruses or bacteria. The final goal of the bundle is to provide tools and resources to collect and analyse data on new pathogens (bacteria and viruses) and their relation to hosts (humans, animals, plants).
- Published
- 2021
19. Milder presentation of TELO2-related syndrome in two sisters homozygous for the p.Arg609His pathogenic variant
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Francesco Musacchia, Valentina Duga, Antonietta Coppola, Valeria Capra, Corrado Romano, Diego Carrella, Annalaura Torella, Margherita Mutarelli, Stefano D'Arrigo, Martino Montomoli, Michele Pinelli, Nicola Brunetti-Pierri, Vincenzo Nigro, Gerarda Cappuccio, Chiara Pantaleoni, Raffaele Castello, Luisa Chiapparini, Claudia Ciaccio, Sandro Banfi, Albina Tummolo, Silvia Maitz, Daniele De Brasi, Isabella Moroni, Giuseppina Vitiello, Vincenzo Leuzzi, Silvia Esposito, Donatella Milani, Marcella Zollino, Giancarlo Parenti, Claudia Santoro, Angelo Selicorni, Ciaccio, C., Duga, V., Pantaleoni, C., Esposito, S., Moroni, I., Pinelli, M., Castello, R., Nigro, V., Chiapparini, L., D'Arrigo, S., Torella, A., Cappuccio, G., Musacchia, F., Mutarelli, M., Carrella, D., Vitiello, G., Parenti, G., Capra, V., Leuzzi, V., Selicorni, A., Maitz, S., Brunetti-Pierri, N., Banfi, S., Zollino, M., Montomoli, M., Milani, D., Romano, C., Tummolo, A., De Brasi, D., Coppola, A., and Santoro, C.
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Microcephaly ,Ataxia ,Adolescent ,Developmental Disabilitie ,Developmental Disabilities ,Mutation, Missense ,Intellectual disability ,Disease ,Compound heterozygosity ,Settore MED/03 - GENETICA MEDICA ,Short stature ,Young Adult ,Genetics ,Humans ,Medicine ,Abnormalities, Multiple ,Global developmental delay ,Genetics (clinical) ,Loss function ,DNA-Repair ,business.industry ,Homozygote ,Syndrome ,General Medicine ,medicine.disease ,Pedigree ,Phenotype ,Female ,medicine.symptom ,TELO2 ,business ,Human - Abstract
Biallelic loss of function of TELO2 gene cause a severe syndromic disease mainly characterized by global developmental delay with poor motor and language acquisitions, microcephaly, short stature, minor facial and limbs anomalies, sleep disorder, spasticity, and balance impairment up to ataxia. TELO2-related syndrome, also known as You-Hoover-Fong Syndrome, is extremely rare and since its first description in 2016 only 8 individuals have been reported, all showing a severe disability. The causative gene is member of the big molecular family of genes responsible for cells proliferation and DNA stability. We describe the case of two sisters, carrying the homozygous p. Arg609His variant of the gene, who present a milder phenotype of TELO2-related syndrome. Such variant has been reported once in a more severely affected patient, in compound heterozygous state associated with the p. Pro260Leu variant, suggesting a possible role of the p. Arg609His variant in determining milder phenotypes. Comparing the siblings with all previously reported cases, we offer an overview on the condition and discuss TELO2 genetic interactions, in order to further explore the molecular bases of this recently described disorder.
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- 2021
20. YAP contributes to DNA methylation remodeling upon mouse embryonic stem cell differentiation
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Fabiana Passaro, Federico Zambelli, Matteo Barbato, Giorgia Di Benedetto, Silvia Parisi, Tommaso Russo, Davide Cacchiarelli, Anna Maria D'Erchia, Dario Antonini, Caterina Manzari, Margherita Mutarelli, Graziano Pesole, Ilaria De Martino, Passaro, Fabiana, De Martino, Ilaria, Zambelli, Federico, Di Benedetto, Giorgia, Barbato, Matteo, D'Erchia, Anna Maria, Manzari, Caterina, Pesole, Graziano, Mutarelli, Margherita, Cacchiarelli, Davide, Antonini, Dario, Parisi, Silvia, and Russo, Tommaso
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0301 basic medicine ,Cellular differentiation ,lncRNA, long noncoding ,Cellular homeostasis ,WWTR1 ,Yes-associated protein ,Biochemistry ,Mice ,Dnmt3l ,DMRs, differentially methylated regions ,FACS, fluorescence-activated cell sorting ,DNA (Cytosine-5-)-Methyltransferases ,CTR KD, control KD ,LIF, leukemia inhibitory factor ,Cell Differentiation ,Mouse Embryonic Stem Cells ,DNA methyltransferases ,yes-associated protein (YAP) ,differentiation ,embryonic stem cells ,TEADs, Transcriptional Enhanced Associate Domains ,Cell biology ,SFEBs, serum-free embryoid bodies ,AP, alkaline phosphatase ,DNA methylation ,embryonic structures ,Signal Transduction ,Research Article ,ChIP-Seq, chromatin immunoprecipitation sequencing ,Biology ,03 medical and health sciences ,stemness ,Animals ,Epigenetics ,Molecular Biology ,Transcription factor ,Adaptor Proteins, Signal Transducing ,GO, gene ontology ,ephemeron ,KD, knockdown ,Hippo signaling pathway ,KO, knockout ,030102 biochemistry & molecular biology ,YAP-Signaling Proteins ,Cell Biology ,ESCs, embryonic stem cells ,DNA Methylation ,FC, fold change ,pluripotency ,TAZ, WW-domain-containing transcription regulator 1 (WWTR1 also known as TAZ) ,embryonic stem cell ,YAP, Yes-associated protein ,030104 developmental biology ,epiblast-like stem cells ,Histone deacetylase complex ,OE, overexpression - Abstract
The Yes-associated protein (YAP), one of the major effectors of the Hippo pathway together with its related protein WW-domain-containing transcription regulator 1 (WWTR1; also known as TAZ), mediates a range of cellular processes from proliferation and death to morphogenesis. YAP and WW-domain-containing transcription regulator 1 (WWTR1; also known as TAZ) regulate a large number of target genes, acting as coactivators of DNA-binding transcription factors or as negative regulators of transcription by interacting with the nucleosome remodeling and histone deacetylase complexes. YAP is expressed in self-renewing embryonic stem cells (ESCs), although it is still debated whether it plays any crucial roles in the control of either stemness or differentiation. Here we show that the transient downregulation of YAP in mouse ESCs perturbs cellular homeostasis, leading to the inability to differentiate properly. Bisulfite genomic sequencing revealed that this transient knockdown caused a genome-wide alteration of the DNA methylation remodeling that takes place during the early steps of differentiation, suggesting that the phenotype we observed might be due to the dysregulation of some of the mechanisms involved in regulation of ESC exit from pluripotency. By gene expression analysis, we identified two molecules that could have a role in the altered genome-wide methylation profile: the long noncoding RNA ephemeron, whose rapid upregulation is crucial for the transition of ESCs into epiblast, and the methyltransferase-like protein Dnmt3l, which, during the embryo development, cooperates with Dnmt3a and Dnmt3b to contribute to the de novo DNA methylation that governs early steps of ESC differentiation. These data suggest a new role for YAP in the governance of the epigenetic dynamics of exit from pluripotency.
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- 2020
21. Altered
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Luca, Parrillo, Rosa, Spinelli, Michele, Longo, Antonella, Desiderio, Paola, Mirra, Cecilia, Nigro, Francesca, Fiory, Shahram, Hedjazifar, Margherita, Mutarelli, Annamaria, Carissimo, Pietro, Formisano, Claudia, Miele, Ulf, Smith, Gregory Alexander, Raciti, and Francesco, Beguinot
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Adult ,Male ,Mice ,Adipogenesis ,Diabetes Mellitus, Type 2 ,3T3-L1 Cells ,Receptor-Like Protein Tyrosine Phosphatases, Class 2 ,Animals ,Humans ,Female ,DNA Methylation ,Epigenesis, Genetic - Published
- 2020
22. TFEB regulates murine liver cell fate during development and regeneration
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Tuong Huynh, Alessia Calcagni, Ayrea Hurley, Milton J. Finegold, Stuart J. Forbes, Nunzia Pastore, William R. Lagor, Luca D’Orsi, Carmine Settembre, Niculin J. Herz, Marco De Giorgi, Tiemo J. Klisch, Lorenzo Brunetti, David D. Moore, Margherita Mutarelli, Andrea Ballabio, Niya Aleksieva, Annamaria Carissimo, Kangho Ho Kim, Pastore, N., Huynh, T., Herz, N. J., Calcagni', A., Klisch, T. J., Brunetti, L., Kim, K. H., De Giorgi, M., Hurley, A., Carissimo, A., Mutarelli, M., Aleksieva, N., D'Orsi, L., Lagor, W. R., Moore, D. D., Settembre, C., Finegold, M. J., Forbes, S. J., and Ballabio, A.
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0301 basic medicine ,Cellular differentiation ,General Physics and Astronomy ,Regenerative Medicine ,Inbred C57BL ,Oral and gastrointestinal ,Transgenic ,Cholangiocarcinoma ,Mice ,0302 clinical medicine ,Stem Cell Research - Nonembryonic - Human ,Models ,2.1 Biological and endogenous factors ,Aetiology ,Promoter Regions, Genetic ,Induced pluripotent stem cell ,lcsh:Science ,Cancer ,Pediatric ,Hepatocyte differentiation ,Transdifferentiation ,Multidisciplinary ,Basic Helix-Loop-Helix Leucine Zipper Transcription Factors ,Liver Disease ,Liver cell ,Stem Cells ,Cell Differentiation ,SOX9 Transcription Factor ,Cell biology ,Up-Regulation ,Phenotype ,Liver ,030220 oncology & carcinogenesis ,Stem Cell Research - Nonembryonic - Non-Human ,Stem cell ,Protein Binding ,Liver Cancer ,1.1 Normal biological development and functioning ,Science ,Chronic Liver Disease and Cirrhosis ,Down-Regulation ,Mice, Transgenic ,Biology ,Cell fate determination ,Models, Biological ,Article ,General Biochemistry, Genetics and Molecular Biology ,Cholangiocyte ,Promoter Regions ,03 medical and health sciences ,Rare Diseases ,Genetic ,Underpinning research ,Spheroids, Cellular ,Genetics ,Animals ,Regeneration ,Cell Lineage ,Cell Proliferation ,General Chemistry ,Stem Cell Research ,Biological ,Mice, Inbred C57BL ,030104 developmental biology ,Bile Duct Neoplasms ,Bile Ducts ,Hepatocytes ,TFEB ,lcsh:Q ,Cellular ,Spheroids ,Digestive Diseases - Abstract
It is well established that pluripotent stem cells in fetal and postnatal liver (LPCs) can differentiate into both hepatocytes and cholangiocytes. However, the signaling pathways implicated in the differentiation of LPCs are still incompletely understood. Transcription Factor EB (TFEB), a master regulator of lysosomal biogenesis and autophagy, is known to be involved in osteoblast and myeloid differentiation, but its role in lineage commitment in the liver has not been investigated. Here we show that during development and upon regeneration TFEB drives the differentiation status of murine LPCs into the progenitor/cholangiocyte lineage while inhibiting hepatocyte differentiation. Genetic interaction studies show that Sox9, a marker of precursor and biliary cells, is a direct transcriptional target of TFEB and a primary mediator of its effects on liver cell fate. In summary, our findings identify an unexplored pathway that controls liver cell lineage commitment and whose dysregulation may play a role in biliary cancer., The Transcription Factor EB (TFEB) is known to regulate cellular homeostasis and energy metabolism, but its role in cell fate determination in the liver is unknown. Here, the authors show that TFEB regulates the progenitor/cholangiocyte lineage and that its depletion prevents tissue recovery upon injury.
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- 2020
23. De novo SMARCA2 variants clustered outside the helicase domain cause a new recognizable syndrome with intellectual disability and blepharophimosis distinct from Nicolaides-Baraitser syndrome
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Gerarda Cappuccio, Camille Sayou, Pauline Le Tanno, Emilie Tisserant, Ange-Line Bruel, Sara El Kennani, Joaquim Sá, Karen J. Low, Cristina Dias, Markéta Havlovicová, Miroslava Hančárová, Evan E. Eichler, Françoise Devillard, Sébastien Moutton, Julien Van-Gils, Christèle Dubourg, Sylvie Odent, Bénédicte Gerard, Amélie Piton, Toshiyuki Yamamoto, Nobuhiko Okamoto, Helen Firth, Kay Metcalfe, Anna Moh, Kimberly A. Chapman, Erfan Aref-Eshghi, Jennifer Kerkhof, Annalaura Torella, Vincenzo Nigro, Laurence Perrin, Juliette Piard, Gwenaël Le Guyader, Thibaud Jouan, Christel Thauvin-Robinet, Yannis Duffourd, Jaya K. George-Abraham, Catherine A. Buchanan, Denise Williams, Usha Kini, Kate Wilson, Nicola Brunetti-Pierri, Giorgio Casari, Michele Pinelli, Francesco Musacchia, Margherita Mutarelli, Diego Carrella, Giuseppina Vitiello, Valeria Capra, Giancarlo Parenti, Vincenzo Leuzzi, Angelo Selicorni, Silvia Maitz, Sandro Banfi, Marcella Zollino, Mario Montomoli, Donatelli Milani, Corrado Romano, Albina Tummolo, Daniele De Brasi, Antonietta Coppola, Claudia Santoro, Angela Peron, Chiara Pantaleoni, Raffaele Castello, Stefano D’Arrigo, Sérgio B. Sousa, Raoul C.M. Hennekam, Bekim Sadikovic, Julien Thevenon, Jérôme Govin, Antonio Vitobello, Università degli studi di Napoli Federico II, Institute for Advanced Biosciences / Institut pour l'Avancée des Biosciences (Grenoble) (IAB), Centre Hospitalier Universitaire [Grenoble] (CHU)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Etablissement français du sang - Auvergne-Rhône-Alpes (EFS)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes (UGA), Lipides - Nutrition - Cancer [Dijon - U1231] (LNC), Université de Bourgogne (UB)-AgroSup Dijon - Institut National Supérieur des Sciences Agronomiques, de l'Alimentation et de l'Environnement-Institut National de la Santé et de la Recherche Médicale (INSERM), University Hospital Motol [Prague], University of Washington [Seattle], Centre Hospitalier Universitaire [Grenoble] (CHU), Université de Bordeaux (UB), Institut de Génétique et Développement de Rennes (IGDR), Structure Fédérative de Recherche en Biologie et Santé de Rennes ( Biosit : Biologie - Santé - Innovation Technologique )-Centre National de la Recherche Scientifique (CNRS)-Université de Rennes 1 (UR1), Université de Rennes (UNIV-RENNES)-Université de Rennes (UNIV-RENNES), Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), Université de Strasbourg (UNISTRA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), London Health Sciences Center (LHSC), Université de Franche-Comté (UFC), Université Bourgogne Franche-Comté [COMUE] (UBFC), Equipe GAD (LNC - U1231), Université de Bourgogne (UB)-AgroSup Dijon - Institut National Supérieur des Sciences Agronomiques, de l'Alimentation et de l'Environnement-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Bourgogne (UB)-AgroSup Dijon - Institut National Supérieur des Sciences Agronomiques, de l'Alimentation et de l'Environnement-Institut National de la Santé et de la Recherche Médicale (INSERM), Centre Hospitalier Universitaire de Dijon - Hôpital François Mitterrand (CHU Dijon), Academic Medical Center - Academisch Medisch Centrum [Amsterdam] (AMC), University of Amsterdam [Amsterdam] (UvA), Centre de génétique - Centre de référence des maladies rares, anomalies du développement et syndromes malformatifs (CHU de Dijon), GSP15001, Fondazione Telethon, 209568/Z/17/Z, Wellcome Trust, Cappuccio, G., Sayou, C., Tanno, P. L., Tisserant, E., Bruel, A. -L., Kennani, S. E., Sa, J., Low, K. J., Dias, C., Havlovicova, M., Hancarova, M., Eichler, E. E., Devillard, F., Moutton, S., Van-Gils, J., Dubourg, C., Odent, S., Gerard, B., Piton, A., Yamamoto, T., Okamoto, N., Firth, H., Metcalfe, K., Moh, A., Chapman, K. A., Aref-Eshghi, E., Kerkhof, J., Torella, A., Nigro, V., Perrin, L., Piard, J., Le Guyader, G., Jouan, T., Thauvin-Robinet, C., Duffourd, Y., George-Abraham, J. K., Buchanan, C. A., Williams, D., Kini, U., Wilson, K., Brunetti-Pierri, N., Casari, G., Pinelli, M., Musacchia, F., Mutarelli, M., Carrella, D., Vitiello, G., Capra, V., Parenti, G., Leuzzi, V., Selicorni, A., Maitz, S., Banfi, S., Zollino, M., Montomoli, M., Milani, D., Romano, C., Tummolo, A., De Brasi, D., Coppola, A., Santoro, C., Peron, A., Pantaleoni, C., Castello, R., D'Arrigo, S., Sousa, S. B., Hennekam, R. C. M., Sadikovic, B., Thevenon, J., Govin, J., Vitobello, A., University of Naples Federico II = Università degli studi di Napoli Federico II, Université de Bourgogne (UB)-Institut National de la Santé et de la Recherche Médicale (INSERM)-AgroSup Dijon - Institut National Supérieur des Sciences Agronomiques, de l'Alimentation et de l'Environnement, Université de Rennes (UR)-Centre National de la Recherche Scientifique (CNRS)-Structure Fédérative de Recherche en Biologie et Santé de Rennes ( Biosit : Biologie - Santé - Innovation Technologique ), Université de Bourgogne (UB)-Institut National de la Santé et de la Recherche Médicale (INSERM)-AgroSup Dijon - Institut National Supérieur des Sciences Agronomiques, de l'Alimentation et de l'Environnement-Université de Bourgogne (UB)-Institut National de la Santé et de la Recherche Médicale (INSERM)-AgroSup Dijon - Institut National Supérieur des Sciences Agronomiques, de l'Alimentation et de l'Environnement, General Paediatrics, APH - Quality of Care, and Brunetti-Pierri, Nicola
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Foot Deformities ,Foot Deformities, Congenital ,[SDV]Life Sciences [q-bio] ,Biology ,Blepharophimosis ,Settore MED/03 - GENETICA MEDICA ,Hypotrichosis ,Chromatin remodeling ,03 medical and health sciences ,Congenital ,0302 clinical medicine ,Neurodevelopmental disorder ,Intellectual Disability ,Intellectual disability ,SMARCA2 ,medicine ,Humans ,Gene ,Genetics (clinical) ,030304 developmental biology ,Genetics ,0303 health sciences ,BIS ,Facies ,medicine.disease ,Phenotype ,neurodevelopmental disorder ,Nicolaides–Baraitser syndrome ,intellectual disability ,DNA methylation ,030217 neurology & neurosurgery ,Transcription Factors - Abstract
International audience; Purpose: Nontruncating variants in SMARCA2, encoding a catalytic subunit of SWI/SNF chromatin remodeling complex, cause Nicolaides-Baraitser syndrome (NCBRS), a condition with intellectual disability and multiple congenital anomalies. Other disorders due to SMARCA2 are unknown.Methods: By next-generation sequencing, we identified candidate variants in SMARCA2 in 20 individuals from 18 families with a syndromic neurodevelopmental disorder not consistent with NCBRS. To stratify variant interpretation, we functionally analyzed SMARCA2 variants in yeasts and performed transcriptomic and genome methylation analyses on blood leukocytes.Results: Of 20 individuals, 14 showed a recognizable phenotype with recurrent features including epicanthal folds, blepharophimosis, and downturned nasal tip along with variable degree of intellectual disability (or blepharophimosis intellectual disability syndrome [BIS]). In contrast to most NCBRS variants, all SMARCA2 variants associated with BIS are localized outside the helicase domains. Yeast phenotype assays differentiated NCBRS from non-NCBRS SMARCA2 variants. Transcriptomic and DNA methylation signatures differentiated NCBRS from BIS and those with nonspecific phenotype. In the remaining six individuals with nonspecific dysmorphic features, clinical and molecular data did not permit variant reclassification.Conclusion: We identified a novel recognizable syndrome named BIS associated with clustered de novo SMARCA2 variants outside the helicase domains, phenotypically and molecularly distinct from NCBRS.
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- 2020
24. Altered PTPRD DNA Methylation Associates With Restricted Adipogenesis in Healthy First-Degree Relatives of Type 2 Diabetes Subjects
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Paola Mirra, Ulf Smith, Francesca Fiory, Annamaria Carissimo, Gregory Alexander Raciti, Antonella Desiderio, Francesco Beguinot, Rosa Spinelli, Shahram Hedjazifar, Luca Parrillo, Cecilia Nigro, Michele Longo, Claudia Miele, Pietro Formisano, Margherita Mutarelli, Parrillo, Luca, Spinelli, Rosa, Longo, Michele, Desiderio, Antonella, Mirra, Paola, Nigro, Cecilia, Fiory, Francesca, Hedjazifar, Shahram, Mutarelli, Margherita, Carissimo, Annamaria, Formisano, Pietro, Miele, Claudia, Smith, Ulf, Raciti, Gregory Alexander, and Beguinot, Francesco
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0301 basic medicine ,Genetics ,Cancer Research ,DNA methylation ,Bisulfite sequencing ,030209 endocrinology & metabolism ,Methylation ,Biology ,PTPRD Gene ,Type 2 diabete ,adipose tissue ,03 medical and health sciences ,030104 developmental biology ,0302 clinical medicine ,Differentially methylated regions ,Adipogenesis ,pre-adipocyte ,gene expression ,Epigenetics ,Methylated DNA immunoprecipitation ,Type 2 diabetes ,epigenetics ,epigenetic - Abstract
Aim: First-degree relatives (FDR) of individuals with Type 2 diabetes (T2D) feature restricted adipogenesis, which render them more vulnerable to T2D. Epigenetics may contribute to these abnormalities. Methods: FDR pre-adipocyte Methylome and Transcriptome were investigated by MeDIP- and RNA-Seq, respectively. Results: Methylome analysis revealed 2841 differentially methylated regions (DMR) in FDR. Most DMR localized into gene-body and were hypomethylated. The strongest hypomethylation signal was identified in an intronic-DMR at the PTPRD gene. PTPRD hypomethylation in FDR was confirmed by bisulphite sequencing and was responsible for its upregulation. Interestingly, Ptprd-overexpression in 3T3-L1 pre-adipocytes inhibited adipogenesis. Notably, the validated PTPRD-associated DMR was significantly hypomethylated in peripheral blood leukocytes from the same FDR individuals. Finally, PTPRD methylation pattern was also replicated in obese individuals. Conclusion: Our findings indicated a previously unrecognized role of PTPRD in restraining adipogenesis. This abnormality may contribute to increase FDR proclivity toward T2D.
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- 2020
25. Esrrb Conveys Naïve Pluripotent Cells Through The Formative Transcriptional Program
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Cristina Malucelli, Jamie A. Hackett, Margherita Mutarelli, Annamaria Carissimo, Francesco Panariello, Valentina Perrera, Graziano Martello, Elena Carbognin, Valentina Carlini, Marcella Cesana, and Davide Cacchiarelli
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medicine.anatomical_structure ,Somatic cell ,Epiblast ,education ,Cell ,medicine ,Gene regulatory network ,Embryo ,Cell fate determination ,Biology ,Stem cell ,Embryonic stem cell ,Cell biology - Abstract
Pluripotency is the potential of a single cell to give rise to all embryonic lineages and first emerges in the naive epiblast of the preimplantation embryo. It has been proposed that upon implantation, epiblast cells transit to a formative phase, which is preparatory for their differentiation into all somatic lineages and primordial germ cells (PGCs). Murine naive embryonic stem cells (ESCs) recapitulate the molecular and functional properties of the naive epiblast, including the capacity to acquire a formative state and differentiate. However, the network of regulators and functional relevance of formative transition remain unresolved. Here we observe that differentiating ESCs transiently activate a distinct transcriptional program consistent with a formative state, after whose completion cells are irreversibly committed to differentiate. To our surprise, we observed that Esrrb, a pivotal naive pluripotency factor, is both sufficient and required to activate the formative program. Mechanistically, in naive cells ESRRB occupies both naive and formative gene loci. During formative transition ESRRB binding at naive genes is lost, while binding on formative genes is consolidated. Finally, when both naive and formative transcriptional programs are inactivated, ESRRB occupancy is mostly lost and cells irreversibly commit to differentiate. Genetic inactivation of Esrrb leads to failure to induce the appropriate formative program, which functionally results in severely impaired PGC specification and accelerated spontaneous mesendoderm differentiation. Thus, Esrrb is critical for activating the formative transition and consequently for executing timely and unbiased multilineage differentiation of murine pluripotent cells. We propose that similar in-built differentiation circuits might be found in other species and stem cell types.
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- 2020
26. BATS: a Bayesian user-friendly software for Analyzing Time Series microarray experiments.
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Claudia Angelini, Luisa Cutillo, Daniela De Canditiis, Margherita Mutarelli, and Marianna Pensky
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- 2008
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27. Time-course analysis of genome-wide gene expression data from hormone-responsive human breast cancer cells.
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Margherita Mutarelli, Luigi Cicatiello, Lorenzo Ferraro, Olì M. V. Grober, Maria Ravo, Angelo M. Facchiano, Claudia Angelini, and Alessandro Weisz
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- 2008
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28. Microdeletion of pseudogene chr14.232.a affects LRFN5 expression in cells of a patient with autism spectrum disorder
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Annalisa Esposito, Marianna Alagia, Roberta Borzone, Floriana Imperati, Sergio Attanasio, Nicola Brunetti-Pierri, Angela Mormile, Margherita Mutarelli, Ennio Del Giudice, Sandro Banfi, Lucio Nitsch, Rita Genesio, Gerarda Cappuccio, Marianthi Karali, Cappuccio, G., Attanasio, S., Alagia, M., Mutarelli, M., Borzone, R., Karali, M., Genesio, R., Mormile, A., Nitsch, L., Imperati, F., Esposito, A., Banfi, S., Del Giudice, E., Brunetti-Pierri, N., Alagia, Marianna, and Borzone, Roberta
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Proband ,Male ,Adolescent ,Autism Spectrum Disorder ,Pseudogene ,Gene Expression ,Biology ,Brief Communication ,Pathogenesis ,03 medical and health sciences ,Plasmid ,Exome Sequencing ,Genetics ,medicine ,Humans ,Genetic Predisposition to Disease ,Gene ,Genetics (clinical) ,Skin ,Chromosomes, Human, Pair 14 ,0303 health sciences ,Membrane Glycoproteins ,030305 genetics & heredity ,RNA ,Transfection ,Fibroblasts ,medicine.disease ,Magnetic Resonance Imaging ,Autism spectrum disorder ,Chromosome Deletion ,Pseudogenes - Abstract
We identified a 14q21.2 microdeletion in a 16-year-old boy with autism spectrum disorder (ASD), IQ in the lower part of normal range but high-functioning memory skills. The deletion affects a gene desert, and the non-deletedgene closest to the microdeletion boundaries is LRFN5, which encodes a protein involved in synaptic plasticity and implicated in neuro-psychiatric disorders. LRFN5 expression was significantly decreased in the proband’s skin fibroblasts. The deleted region includes the pseudogene chr14.232.a, which is transcribed into a long non-coding RNA (lncLRFN5-10), whose levels were also significantly reduced in the proband’s fibroblasts compared to controls. Transfection of the patient’s fibroblasts with a plasmid expressing chr14.232.a significantly increased LRFN5 expression, while siRNA targeting chr14.232.a-derived lncLRFN5-10 reduced LRFN5 levels. In summary, we report on an individual with ASD carrying a microdeletion encompassing the pseudogene chr14.232.a encoding for lncLRFN5-10, which was found to affect the expression levels of the nearby, non-deleted LRFN5. This case illustrates the potential role of long non-coding RNAs in regulating expression of neighbouring genes with a functional role in ASD pathogenesis.
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- 2019
29. Nutrient-sensitive transcription factors TFEB and TFE3 couple autophagy and metabolism to the peripheral clock
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Kenichiro Kinouchi, Anna Vainshtein, Patrizia Annunziata, Tuong Huynh, Nicola Brunetti-Pierri, Paolo Sassone-Corsi, Lorenzo Brunetti, Andrea Ballabio, Tiemo J. Klisch, Niculin J. Herz, Nunzia Pastore, Margherita Mutarelli, Pastore, N., Vainshtein, A., Herz, N. J., Huynh, T., Brunetti, L., Klisch, T. J., Mutarelli, M., Annunziata, P., Kinouchi, K., Brunetti-Pierri, N., Sassone-Corsi, P., and Ballabio, A.
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MiT‐TFE ,Male ,Circadian clock ,Inbred C57BL ,Medical and Health Sciences ,Transgenic ,Mice ,0302 clinical medicine ,Group D ,Gene expression ,News & Views ,Cells, Cultured ,Regulation of gene expression ,0303 health sciences ,Cultured ,Basic Helix-Loop-Helix Leucine Zipper Transcription Factors ,General Neuroscience ,REV-ERB alpha ,Articles ,Biological Sciences ,Cell biology ,Circadian Rhythm ,Crosstalk (biology) ,circadian rhythm ,gene oscillation ,MiT-TFE ,REV-ERBα ,Animals ,Binding Sites ,Gene Expression Regulation ,HEK293 Cells ,Humans ,Mice, Inbred C57BL ,Mice, Transgenic ,Nuclear Receptor Subfamily 1, Group D, Member 1 ,Nutrients ,Transcription Factors ,Autophagy ,Circadian Clocks ,Energy Metabolism ,Sleep Research ,REV‐ERBα ,Member 1 ,Nuclear Receptor Subfamily 1 ,1.1 Normal biological development and functioning ,Cells ,Biology ,General Biochemistry, Genetics and Molecular Biology ,03 medical and health sciences ,Underpinning research ,Information and Computing Sciences ,Genetics ,Circadian rhythm ,Molecular Biology ,Transcription factor ,030304 developmental biology ,Nutrition ,General Immunology and Microbiology ,Neurosciences ,TFEB ,030217 neurology & neurosurgery ,Developmental Biology - Abstract
Autophagy and energy metabolism are known to follow a circadian pattern. However, it is unclear whether autophagy and the circadian clock are coordinated by common control mechanisms. Here, we show that the oscillation of autophagy genes is dependent on the nutrient-sensitive activation of TFEB and TFE3, key regulators of autophagy, lysosomal biogenesis, and cell homeostasis. TFEB and TFE3 display a circadian activation over the 24-h cycle and are responsible for the rhythmic induction of genes involved in autophagy during the light phase. Genetic ablation of TFEB and TFE3 in mice results in deregulated autophagy over the diurnal cycle and altered gene expression causing abnormal circadian wheel-running behavior. In addition, TFEB and TFE3 directly regulate the expression of Rev-erbα (Nr1d1), a transcriptional repressor component of the core clock machinery also involved in the regulation of whole-body metabolism and autophagy. Comparative analysis of the cistromes of TFEB/TFE3 and REV-ERBα showed an extensive overlap of their binding sites, particularly in genes involved in autophagy and metabolic functions. These data reveal a direct link between nutrient and clock-dependent regulation of gene expression shedding a new light on the crosstalk between autophagy, metabolism, and circadian cycles.
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- 2018
30. Expanding the phenotype of RTTN variations: a new family with primary microcephaly, severe growth failure, brain malformations and dermatitis
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Adalgisa Festa, Giulio Piluso, Vincenzo Nigro, Annalaura Torella, Margherita Mutarelli, F. Del Vecchio Blanco, Carlo Capristo, E. Miraglia del Giudice, Mario Cirillo, Laura Perrone, Teresa Giugliano, Pierluigi Marzuillo, Claudia Santoro, Anna Grandone, and Grazia Cirillo
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0301 basic medicine ,Genetics ,Lissencephaly ,030105 genetics & heredity ,Biology ,medicine.disease ,Developmental disorder ,03 medical and health sciences ,Exon ,030104 developmental biology ,Failure to thrive ,medicine ,Polymicrogyria ,Missense mutation ,medicine.symptom ,Primordial dwarfism ,Genetics (clinical) ,Exome sequencing - Abstract
Primary autosomal recessive microcephaly (MCPH) is a developmental disorder characterized by prenatal onset of abnormal brain growth. MCPH occurs both alone and as part of a broad range of neurodevelopmental syndromes with or without cortical malformations and growth retardation. Here we report a consanguineous Moroccan family with two siblings affected by severe primary microcephaly, failure to thrive, congenital dermatitis and severe developmental delay. Brain magnetic resonance imaging showed lissencephaly of frontal lobes and periventricular heterotopia of the gray matter. We performed both Comparative Genomic Hybridization array and whole exome sequencing (WES) analyses of the kindred. No quantitative defects were detected. However, WES identified a new homozygous missense variation in the penultimate nucleotide of exon 23 of RTTN gene (c.2953A>G;pArg985Gly). cDNA sequencing revealed two abnormal spliced products, one lacking only exon 23 and the other lacking exons 22 and 23 (out-of-frame). RTTN is a protein involved in cilia structure and function. Homozygous mutations in RTTN gene have been described in bilateral diffuse isolated polymicrogyria and, more recently, in microcephalic primordial dwarfism (PD). We found a novel homozygous mutation in RTTN associated with microcephalic PD as well as complex brain malformations and congenital dermatitis, thus expanding the phenotypic spectrum of both RTTN-associated diseases and ciliary dysfunction.
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- 2016
31. High-resolution analysis of the human retina miRNome reveals isomiR variations and novel microRNAs
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Diego Carrella, Concetta Ambrosio, Marianthi Karali, Maria Persico, Vincenzo Nigro, Annamaria Carissimo, Veer Singh Marwah, Sandro Banfi, Michele Pinelli, Diego Ponzin, Mariateresa Pizzo, Diego di Bernardo, Stefano Ferrari, Margherita Mutarelli, Karali, Marianthi, Persico, Maria, Mutarelli, Margherita, Carissimo, Annamaria, Pizzo, Mariateresa, Singh Marwah, Veer, Ambrosio, Concetta, Pinelli, Michele, Carrella, Diego, Ferrari, Stefano, Ponzin, Diego, Nigro, Vincenzo, DI BERNARDO, Diego, Banfi, Sandro, Karali, M, Persico, M, Mutarelli, M, Carissimo, A, Pizzo, M, Marwah, V, Ambrosio, C, Pinelli, M, Carrella, D, Ferrari, S, Ponzin, D, and Di Bernardo, D
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0301 basic medicine ,MOUSE RETINA ,ANIMAL DEVELOPMENT ,SMALL RNAS ,EXPRESSION ,DIFFERENTIATION ,CLUSTER ,IMPACT ,GENES ,INACTIVATION ,DEGENERATION ,Retina homeostasis ,Data Resources and Analyses ,Retinal Pigment Epithelium ,Computational biology ,Biology ,Bioinformatics ,Retina ,Deep sequencing ,Transcriptome ,03 medical and health sciences ,0302 clinical medicine ,IsomiR ,microRNA ,Genetics ,medicine ,Humans ,Retinal pigment epithelium ,High-Throughput Nucleotide Sequencing ,eye diseases ,MicroRNAs ,030104 developmental biology ,medicine.anatomical_structure ,sense organs ,030217 neurology & neurosurgery ,Function (biology) - Abstract
MicroRNAs play a fundamental role in retinal development and function. To characterise the miRNome of the human retina, we carried out deep sequencing analysis on sixteen individuals. We established the catalogue of retina-expressed miRNAs, determined their relative abundance and found that a small number of miRNAs accounts for almost 90% of the retina miRNome. We discovered more than 3000 miRNA variants (isomiRs), encompassing a wide range of sequence variations, which include seed modifications that are predicted to have an impact on miRNA action. We demonstrated that a seed-modifying isomiR of the retina-enriched miR-124-3p was endowed with different targeting properties with respect to the corresponding canonical form. Moreover, we identified 51 putative novel, retina-specific miRNAs and experimentally validated the expression for nine of them. Finally, a parallel analysis of the human Retinal Pigment Epithelium (RPE)/choroid, two tissues that are known to be crucial for retina homeostasis, yielded notably distinct miRNA enrichment patterns compared to the retina. The generated data are accessible through an ad hoc database. This study is the first to reveal the complexity of the human retina miRNome at nucleotide resolution and constitutes a unique resource to assess the contribution of miRNAs to the pathophysiology of the human retina.
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- 2016
32. Direct generation of human naive induced pluripotent stem cells from somatic cells in microfluidics
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Irene Zorzan, Margherita Mutarelli, Chiara Romualdi, Paolo Martini, Marco Pellegrini, Stefano Giulitti, Graziano Martello, Davide Cacchiarelli, Nicola Elvassore, Onelia Gagliano, Michael J. Ziller, Giulitti, Stefano, Pellegrini, Marco, Zorzan, Irene, Martini, Paolo, Gagliano, Onelia, Mutarelli, Margherita, Ziller, Michael Johanne, Cacchiarelli, Davide, Romualdi, Chiara, Elvassore, Nicola, and Martello, Graziano
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Homeobox protein NANOG ,Somatic cell ,Induced Pluripotent Stem Cells ,Karyotype ,Microfluidics ,Kruppel-Like Transcription Factors ,Germ layer ,Biology ,Regenerative Medicine ,Regenerative medicine ,Proto-Oncogene Proteins c-myc ,03 medical and health sciences ,Kruppel-Like Factor 4 ,Mice ,0302 clinical medicine ,SOX2 ,Animals ,Humans ,Epigenetics ,RNA, Messenger ,Induced pluripotent stem cell ,Cells, Cultured ,030304 developmental biology ,0303 health sciences ,SOXB1 Transcription Factors ,Cell Differentiation ,Cell Biology ,3. Good health ,Cell biology ,KLF4 ,030220 oncology & carcinogenesis ,Octamer Transcription Factor-3 ,Germ Layers - Abstract
Induced pluripotent stem cells (iPSCs) are generated via the expression of the transcription factors OCT4 (also known as POU5F1), SOX2, KLF4 and cMYC (OSKM) in somatic cells. In contrast to murine naive iPSCs, conventional human iPSCs are in a more developmentally advanced state called primed pluripotency. Here, we report that human naive iPSCs (niPSCs) can be generated directly from fewer than 1,000 primary human somatic cells, without requiring stable genetic manipulation, via the delivery of modified messenger RNAs using microfluidics. Expression of the OSKM factors in combination with NANOG for 12 days generates niPSCs that are free of transgenes, karyotypically normal and display transcriptional, epigenetic and metabolic features indicative of the naive state. Importantly, niPSCs efficiently differentiate into all three germ layers. While niPSCs can be generated at low frequency under conventional conditions, our microfluidics approach enables the robust and cost-effective production of patient-specific niPSCs for regenerative medicine applications, including disease modelling and drug screening.
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- 2018
33. Lysoplex: An efficient toolkit to detect DNA sequence variations in the autophagy-lysosomal pathway
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Giuseppina Di Fruscio, Andrea Ballabio, Vincenzo Nigro, Thomas Braulke, Angela Schulz, Giancarlo Parenti, Rossella De Cegli, Sandro Banfi, Margherita Mutarelli, Marco Savarese, Di Fruscio, G, Schulz, A, De Cegli, R, Savarese, M, Mutarelli, M, Parenti, Giancarlo, Banfi, S, Braulke, T, Nigro, V, Ballabio, Andrea, Parenti, G, Banfi, Sandro, Nigro, Vincenzo, and Ballabio, A.
- Subjects
autophagy ,WGS, whole genome sequencing ,Endocytic cycle ,Cell ,Biology ,DNA sequencing ,lysosomal storage disorders ,Neuronal Ceroid-Lipofuscinoses ,NCL, neuronal ceroid lipofuscinosis ,autophagy-lysosomal pathway ,medicine ,Humans ,Coding region ,Amino Acid Sequence ,Molecular Biology ,Gene ,Genetics ,ALP, autophagy-lysosomal pathway ,Base Sequence ,Genetic heterogeneity ,genetic variants ,Homozygote ,Autophagy ,DNA ,Cell Biology ,medicine.disease ,3. Good health ,Lysosomal Storage Diseases ,NGS, next-generation sequencing ,medicine.anatomical_structure ,Mutation ,LSDs, lysosomal storage disorders ,next-generation sequencing ,Neuronal ceroid lipofuscinosis ,Clinical Research Paper ,Lysosomes ,WES, whole exome sequencing ,neuronal ceroid lipofuscinoses - Abstract
The autophagy-lysosomal pathway (ALP) regulates cell homeostasis and plays a crucial role in human diseases, such as lysosomal storage disorders (LSDs) and common neurodegenerative diseases. Therefore, the identification of DNA sequence variations in genes involved in this pathway and their association with human diseases would have a significant impact on health. To this aim, we developed Lysoplex, a targeted next-generation sequencing (NGS) approach, which allowed us to obtain a uniform and accurate coding sequence coverage of a comprehensive set of 891 genes involved in lysosomal, endocytic, and autophagic pathways. Lysoplex was successfully validated on 14 different types of LSDs and then used to analyze 48 mutation-unknown patients with a clinical phenotype of neuronal ceroid lipofuscinosis (NCL), a genetically heterogeneous subtype of LSD. Lysoplex allowed us to identify pathogenic mutations in 67% of patients, most of whom had been unsuccessfully analyzed by several sequencing approaches. In addition, in 3 patients, we found potential disease-causing variants in novel NCL candidate genes. We then compared the variant detection power of Lysoplex with data derived from public whole exome sequencing (WES) efforts. On average, a 50% higher number of validated amino acid changes and truncating variations per gene were identified. Overall, we identified 61 truncating sequence variations and 488 missense variations with a high probability to cause loss of function in a total of 316 genes. Interestingly, some loss-of-function variations of genes involved in the ALP pathway were found in homozygosity in the normal population, suggesting that their role is not essential. Thus, Lysoplex provided a comprehensive catalog of sequence variants in ALP genes and allows the assessment of their relevance in cell biology as well as their contribution to human disease.
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- 2015
34. Genetic Association of ARHGAP21 Gene Variant with Mandibular Prognathism
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Vincenzo Nigro, A. Monsurrò, E. Bonci, Letizia Perillo, Annalaura Torella, Margherita Mutarelli, Perillo, Letizia, Monsurrò, A, Bonci, E, Torella, A, Mutarelli, M, and Nigro, Vincenzo
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Adult ,Male ,genetic variant ,Candidate gene ,Adolescent ,Genotype ,Genetic Linkage ,Filamins ,Glycine ,Mutation, Missense ,Bone Morphogenetic Protein 3 ,Penetrance ,Biology ,Young Adult ,Genetic linkage ,Serine ,Humans ,Missense mutation ,Exome ,FLNB ,whole-exome sequencing ,Child ,General Dentistry ,Annexin A2 ,Genetic Association Studies ,Exome sequencing ,Aged ,Genes, Dominant ,Genetic association ,Homeodomain Proteins ,Genetics ,Bone growth ,bone growth ,GTPase-Activating Proteins ,Genetic Variation ,candidate gene ,Sequence Analysis, DNA ,Middle Aged ,Pedigree ,Meiosis ,Malocclusion, Angle Class III ,Prognathism ,Female ,next-generation sequencing ,class III malocclusion - Abstract
Mandibular prognathism (MP) is a recognizable phenotype associated with dentoskeletal class III malocclusion. MP is a complex genetic trait, although familial recurrence also suggests the contribution of single inherited variations. To date, the genetic causes of MP have been investigated using linkage analysis or association studies in pooled families. Here for the first time, next-generation sequencing was used to study a single family with a large number of MP-affected members and to identify MP-related candidate genes. A 6-generation kindred with MP segregating as an autosomal dominant character was recruited. To identify family members affected by MP, a standard cephalometric procedure was used. In 5 MP subjects separated by the largest number of meioses, whole-exome sequencing was performed. Five promising missense gene variants ( BMP3, ANXA2, FLNB, HOXA2, and ARHGAP21) associated with MP were selected and genotyped in most other family members. In this family, MP seemed to consist of 2 distinct genetic branches. Interestingly, the Gly1121Ser variant in the ARHGAP21 gene was found to be shared by all MP individuals in the larger branch of the family with nearly complete penetrance. This variant is rare in the Caucasian population (frequency 0.00034) and is predicted as damaging by all bioinformatic algorithms. ARHGAP21 protein strengthens cell-cell adhesions and may be regulated by bone morphogenetic factors, thus influencing mandibular growth. Further studies in both animal models and human patients are required to clarify the significance of this association.
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- 2015
35. Allelic Expression Imbalance in the Human Retinal Transcriptome and Potential Impact on Inherited Retinal Diseases
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Jan Vetter, Pablo Llavona, Simone Schimpf-Linzenbold, Efdal Yoeruek, Bernd Wissinger, Susanne Kohl, Margherita Mutarelli, Sebastian Thaler, Veer Singh Marwah, Michele Pinelli, and Institute of Biotechnology
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0301 basic medicine ,retina ,lcsh:QH426-470 ,Single-nucleotide polymorphism ,Context (language use) ,Biology ,PHENOTYPE ,expressivity ,Transcriptome ,03 medical and health sciences ,0302 clinical medicine ,Inherited retinal diseases ,Genetics ,3125 Otorhinolaryngology, ophthalmology ,Expressivity (genetics) ,Allele ,penetrance ,MUTATION ,Genetics (clinical) ,Massive parallel sequencing ,Communication ,1184 Genetics, developmental biology, physiology ,DYSTROPHY ,Penetrance ,lcsh:Genetics ,030104 developmental biology ,Expression quantitative trait loci ,030221 ophthalmology & optometry ,allelic expression imbalance - Abstract
Inherited retinal diseases (IRDs) are often associated with variable clinical expressivity (VE) and incomplete penetrance (IP). Underlying mechanisms may include environmental, epigenetic, and genetic factors. Cis-acting expression quantitative trait loci (cis-eQTLs) can be implicated in the regulation of genes by favoring or hampering the expression of one allele over the other. Thus, the presence of such loci elicits allelic expression imbalance (AEI) that can be traced by massive parallel sequencing techniques. In this study, we performed an AEI analysis on RNA-sequencing (RNA-seq) data, from 52 healthy retina donors, that identified 194 imbalanced single nucleotide polymorphisms(SNPs) in 67 IRD genes. Focusing on SNPs displaying AEI at a frequency higher than 10%, we found evidence of AEI in several IRD genes regularly associated with IP and VE (BEST1, RP1, PROM1, and PRPH2). Based on these SNPs commonly undergoing AEI, we performed pyrosequencing in an independent sample set of 17 healthy retina donors in order to confirm our findings. Indeed, we were able to validate CDHR1, BEST1, and PROM1 to be subjected to cis-acting regulation. With this work, we aim to shed light on differentially expressed alleles in the human retina transcriptome that, in the context of autosomal dominant IRD cases, could help to explain IP or VE.
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- 2017
36. Triple Vectors Expand AAV Transfer Capacity in the Retina
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Settimio Rossi, Paola Tiberi, Patrizia Tornabene, Francesca Simonelli, Renato Minopoli, Alberto Auricchio, Andrea Maddalena, Anna Manfredi, Juergen K Naggert, Davide Cacchiarelli, Margherita Mutarelli, Maddalena, Andrea, Tornabene, Patrizia, Tiberi, Paola, Minopoli, Renato, Manfredi, Anna, Mutarelli, Margherita, Rossi, Settimio, Simonelli, Francesca, Naggert, Jurgen K., Cacchiarelli, Davide, and Auricchio, Alberto
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0301 basic medicine ,retina ,Transcription, Genetic ,Swine ,Usher syndrome ,Genetic enhancement ,viruses ,Gene Expression ,law.invention ,Transduction (genetics) ,chemistry.chemical_compound ,Mice ,CDH23 ,law ,Genes, Reporter ,Transduction, Genetic ,Drug Discovery ,Transgenes ,Mice, Knockout ,Recombination, Genetic ,Gene Transfer Techniques ,AAV ,Dependovirus ,Cadherins ,gene therapy ,3. Good health ,Cell biology ,medicine.anatomical_structure ,Recombinant DNA ,Molecular Medicine ,Original Article ,large gene ,Gene Expression Regulation, Viral ,Genetic Vectors ,triple AAV ,Biology ,03 medical and health sciences ,Genetic ,Genetics ,medicine ,Animals ,Humans ,Molecular Biology ,Pharmacology ,Retina ,Drug Discovery3003 Pharmaceutical Science ,Retinal ,Genetic Therapy ,medicine.disease ,030104 developmental biology ,HEK293 Cells ,chemistry ,Alström syndrome - Abstract
Retinal gene transfer with adeno-associated viral (AAV) vectors holds great promise for the treatment of inherited retinal degenerations (IRDs). One limit of AAV is its transfer capacity of about 5 kb, which can be expanded to about 9 kb, using dual AAV vectors. This strategy would still not suffice for treatment of IRDs such as Usher syndrome type 1D or Alström syndrome type I (ALMS) due to mutations in CDH23 or ALMS1, respectively. To overcome this limitation, we generated triple AAV vectors, with a maximal transfer capacity of about 14 kb. Transcriptomic analysis following triple AAV transduction showed the expected full-length products along a number of aberrant transcripts. However, only the full-length transcripts are efficiently translated in vivo. We additionally showed that approximately 4% of mouse photoreceptors are transduced by triple AAV vectors and showed correct localization of recombinant ALMS1. The low-photoreceptor transduction levels might justify the modest and transient improvement we observe in the retina of a mouse model of ALMS. However, the levels of transduction mediated by triple AAV vectors in pig retina reached 40% of those observed with single vectors, and this bodes well for further improving the efficiency of triple AAV vectors in the retina., Graphical Abstract, Maddalena et al. show that the limited DNA transfer capacity of adeno-associated viral (AAV) vectors can be expanded up to 14 kb with triple AAV vectors. The authors show triple AAV-mediated expression of both reporter and large therapeutic genes in vitro and in mouse and pig retina.
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- 2017
37. PML-RARA-associated cooperating mutations belong to a transcriptional network that is deregulated in myeloid leukemias
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Francesco Lo-Coco, Vincenzo Belcastro, Gopuraja Dharmalingam, Alicja M. Gruszka, Pier Giuseppe Pelicci, Giorgio E. M. Melloni, Eugenio Scanziani, Diego di Bernardo, Alessandro Brozzi, Laura Riva, Margherita Mutarelli, Chiara Ronchini, Lucilla Luzi, Serena Lavorgna, Alessandro Rambaldi, Vincenzo Rossi, Orietta Spinelli, Andrea Biondi, Ronchini, C, Brozzi, A, Riva, L, Luzi, L, Gruszka, A. M, Melloni, G. E. M, Scanziani, E, Dharmalingam, G, Mutarelli, M, Belcastro, V, Lavorgna, S, Rossi, V, Spinelli, O, Biondi, A, Rambaldi, A, Lo Coco, F, DI BERNARDO, Diego, Pelicci, P. G., Gruszka, A, Melloni, G, Lo-Coco, F, Di Bernardo, D, and Pelicci, P
- Subjects
Myeloid ,0301 basic medicine ,Acute promyelocytic leukemia ,Cancer Research ,Oncogene Proteins, Fusion ,genetic structures ,Carcinogenesis ,Gene regulatory network ,Acute ,Biology ,Fusion gene ,Databases ,Mice ,03 medical and health sciences ,Animals ,Databases, Genetic ,Gene Regulatory Networks ,Humans ,Leukemia, Myeloid ,Leukemia, Myeloid, Acute ,Leukemia, Promyelocytic, Acute ,NIH 3T3 Cells ,Mutation ,Genetic ,hemic and lymphatic diseases ,medicine ,PML-RARA ,Fusion ,neoplasms ,Gene ,Promyelocytic ,Oncogene Proteins ,Genetics ,Leukemia ,Oncogene ,Myeloid leukemia ,Hematology ,medicine.disease ,030104 developmental biology ,medicine.anatomical_structure ,Oncology ,Settore MED/15 - Malattie del Sangue - Abstract
It has been shown that individual acute myeloid leukemia (AML) patients are characterized by one of few initiating DNA mutations and 5-10 cooperating mutations not yet defined among hundreds identified by massive sequencing of AML genomes. We report an in vivo insertional-mutagenesis screen for genes cooperating with one AML initiating mutations (PML-RARA, oncogene of acute promyelocytic leukemia, APL), which allowed identification of hundreds of genetic cooperators. The cooperators are mutated at low frequency in APL or AML patients but are always abnormally expressed in a cohort of 182 APLs and AMLs analyzed. These deregulations appear non-randomly distributed and present in all samples, regardless of their associated genomic mutations. Reverse-engineering approaches showed that these cooperators belong to a single transcriptional gene network, enriched in genes mutated in AMLs, where perturbation of single genes modifies expression of others. Their gene-ontology analysis showed enrichment of genes directly involved in cell proliferation control. Therefore, the pool of PML-RARA cooperating mutations appears large and heterogeneous, but functionally equivalent and deregulated in the majority of APLs and AMLs. Our data suggest that the high heterogeneity of DNA mutations in APLs and AMLs can be reduced to patterns of gene expression deregulation of a single 'mutated' gene network.Leukemia advance online publication, 20 January 2017; doi:10.1038/leu.2016.386.
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- 2017
38. Clinical and Genetic Evaluation of a Cohort of Pediatric Patients with Severe Inherited Retinal Dystrophies
- Author
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Francesca Simonelli, Raffaella Brunetti-Pierri, Mariateresa Pizzo, Giuseppina Di Fruscio, Marianthi Karali, Vincenzo Nigro, Francesco Testa, Mariaelena Filippelli, Valentina Di Iorio, Sandro Banfi, Margherita Mutarelli, Di Iorio, Valentina, Karali, Marianthi, Brunetti-pierri, Raffaella, Filippelli, Mariaelena, Di Fruscio, Giuseppina, Pizzo, Mariateresa, Mutarelli, Margherita, Nigro, Vincenzo, Testa, Francesco, Banfi, Sandro, and Simonelli, Francesca
- Subjects
0301 basic medicine ,Pediatrics ,medicine.medical_specialty ,Genotype-phenotype correlation ,Achromatopsia ,lcsh:QH426-470 ,Genetic enhancement ,inherited retinal dystrophies ,Leber congenital amaurosis ,Leber congenital amaurosi ,Article ,Pathogenesis ,03 medical and health sciences ,Next generation sequencing ,Genotype ,Retinitis pigmentosa ,Genetics ,Medicine ,Inherited retinal dystrophie ,Genetics (clinical) ,Early onset ,Ellipsoid zone ,business.industry ,medicine.disease ,lcsh:Genetics ,030104 developmental biology ,Cohort ,early onset ,next generation sequencing ,genotype-phenotype correlation ,retinitis pigmentosa ,achromatopsia ,ellipsoid zone ,business ,Retinal Dystrophies - Abstract
We performed a clinical and genetic characterization of a pediatric cohort of patients with inherited retinal dystrophy (IRD) to identify the most suitable cases for gene therapy. The cohort comprised 43 patients, aged between 2 and 18 years, with severe isolated IRD at the time of presentation. The ophthalmological characterization also included assessment of the photoreceptor layer integrity in the macular region (ellipsoid zone (EZ) band). In parallel, we carried out a targeted, next-generation sequencing (NGS)-based analysis using a panel that covers over 150 genes with either an established or a candidate role in IRD pathogenesis. Based on the ophthalmological assessment, the cohort was composed of 24 Leber congenital amaurosis, 14 early onset retinitis pigmentosa, and 5 achromatopsia patients. We identified causative mutations in 58.1% of the cases. We also found novel genotype-phenotype correlations in patients harboring mutations in the CEP290 and CNGB3 genes. The EZ band was detectable in 40%of the analyzed cases, also in patients with genotypes usually associated with severe clinical manifestations. This study provides the first detailed clinical-genetic assessment of severe IRDs with infantile onset and lays the foundation of a standardized protocol for the selection of patients that are more likely to benefit from gene replacement therapeutic approaches. We performed a clinical and genetic characterization of a pediatric cohort of patients with inherited retinal dystrophy (IRD) to identify the most suitable cases for gene therapy. The cohort comprised 43 patients, aged between 2 and 18 years, with severe isolated IRD at the time of presentation. The ophthalmological characterization also included assessment of the photoreceptor layer integrity in the macular region (ellipsoid zone (EZ) band). In parallel, we carried out a targeted, next-generation sequencing (NGS)-based analysis using a panel that covers over 150 genes with either an established or a candidate role in IRD pathogenesis. Based on the ophthalmological assessment, the cohort was composed of 24 Leber congenital amaurosis, 14 early onset retinitis pigmentosa, and 5 achromatopsia patients. We identified causative mutations in 58.1% of the cases. We also found novel genotype-phenotype correlations in patients harboring mutations in the CEP290 and CNGB3 genes. The EZ band was detectable in 40% of the analyzed cases, also in patients with genotypes usually associated with severe clinical manifestations. This study provides the first detailed clinical-genetic assessment of severe IRDs with infantile onset and lays the foundation of a standardized protocol for the selection of patients that are more likely to benefit from gene replacement therapeutic approaches.
- Published
- 2017
39. Computational drugs repositioning identifies inhibitors of oncogenic PI3K/AKT/P70S6K-dependent pathways among FDA-approved compounds
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Manuela Iezzi, Federica Papaccio, Diego di Bernardo, Fabrizio Loreni, Alberto Bardelli, Laura Ciolli, Francesco Sirci, Vittorio Enrico Avvedimento, Margherita Mutarelli, Marcella Mottolese, Valentina Aria, Roberta Bosotti, Luca Cardone, Carla Azzurra Amoreo, Antonella Isacchi, Rosanna Dattilo, Barbara Tumaini, Diego Carrella, Isabella Manni, Carrella, Diego, Manni, Isabella, Tumaini, Barbara, Dattilo, Rosanna, Papaccio, Federica, Mutarelli, Margherita, Sirci, Francesco, Amoreo, Carla A, Mottolese, Marcella, Iezzi, Manuela, Ciolli, Laura, Aria, Valentina, Bosotti, Roberta, Isacchi, Antonella, Loreni, Fabrizio, Bardelli, Alberto, Avvedimento, VITTORIO ENRICO, DI BERNARDO, Diego, and Cardone, Luca
- Subjects
0301 basic medicine ,Veterinary medicine ,Carcinogenesis ,oncogenes ,Ribosomal Protein S6 Kinases 70-kDa ,Phosphatidylinositol 3-Kinases ,Mice ,Drug Approval ,Phosphoinositide-3 Kinase Inhibitors ,Tumor ,Settore BIO/11 ,Ribosomal Protein S6 Kinases, 70-kDa ,Mammary Glands ,gene expression signatures ,Drug repositioning ,Oncology ,PI3K-dependent pathways ,Niclosamide ,Female ,Signal Transduction ,FDA-approved drugs ,drugs network ,Animals ,Antineoplastic Agents ,Breast Neoplasms ,Cell Line ,Humans ,Animal ,Proto-Oncogene Proteins c-akt ,Pyrvinium Compounds ,Computational Biology ,Drug Repositioning ,Mouse Mammary Gland ,gene expression signature ,PI3K-dependent pathway ,03 medical and health sciences ,Mammary Glands, Animal ,P70S6 kinase ,Cell Line, Tumor ,medicine ,Protein kinase B ,PI3K/AKT/mTOR pathway ,FDA-approved drug ,business.industry ,Cancer ,medicine.disease ,030104 developmental biology ,Cancer research ,Breast cancer cells ,business ,Signalling pathways ,Priority Research Paper - Abstract
// Diego Carrella 1 , Isabella Manni 3 , Barbara Tumaini 1 , Rosanna Dattilo 2 , Federica Papaccio 3 , Margherita Mutarelli 1 , Francesco Sirci 1 , Carla A. Amoreo 4 , Marcella Mottolese 4 , Manuela Iezzi 5 , Laura Ciolli 5 , Valentina Aria 6 , Roberta Bosotti 7 , Antonella Isacchi 7 , Fabrizio Loreni 6 , Alberto Bardelli 8,9 , Vittorio E. Avvedimento 10 , Diego di Bernardo 1,11 and Luca Cardone 3 1 Telethon Institute of Genetics and Medicine, Pozzuoli, Naples, Italy 2 Department of Hematology, Oncology and Molecular Medicine, Biobank Unit, Istituto Superiore di Sanita, Rome, Italy 3 Department of Research, Advanced Diagnostics, and Technological Innovations, Regina Elena National Cancer Institute, Rome, Italy 4 S.C. Anatomia Patologica, Regina Elena National Cancer Institute, Rome, Italy 5 Immuno-Oncology Laboratory, Aging Research Center, G. d’Annunzio University of Chieti, Pescara, Italy 6 Department of Biology, University of Rome Tor Vergata, Rome, Italy 7 Nerviano Medical Sciences SRL, Nerviano, Italy 8 Candiolo Cancer Institute-FPO, IRCCS, Candiolo, Torino, Italy 9 Department of Oncology, University of Torino, Candiolo, Torino, Italy 10 Dipartimento di Medicina Molecolare e Biotecnologie Mediche, Universita degli Studi di Napoli “Federico II”, Naples, Italy 11 Department of Electrical Engineering and Information Technology, University of Naples “Federico II”, Naples, Italy Correspondence to: Luca Cardone, email: // Diego di Bernardo, email: // Keywords : oncogenes, PI3K-dependent pathways, gene expression signatures, drugs network, FDA-approved drugs Received : January 03, 2016 Accepted : August 03, 2016 Published : August 16, 2016 Abstract The discovery of inhibitors for oncogenic signalling pathways remains a key focus in modern oncology, based on personalized and targeted therapeutics. Computational drug repurposing via the analysis of FDA-approved drug network is becoming a very effective approach to identify therapeutic opportunities in cancer and other human diseases. Given that gene expression signatures can be associated with specific oncogenic mutations, we tested whether a “reverse” oncogene-specific signature might assist in the computational repositioning of inhibitors of oncogenic pathways. As a proof of principle, we focused on oncogenic PI3K-dependent signalling, a molecular pathway frequently driving cancer progression as well as raising resistance to anticancer-targeted therapies. We show that implementation of “reverse” oncogenic PI3K-dependent transcriptional signatures combined with interrogation of drug networks identified inhibitors of PI3K-dependent signalling among FDA-approved compounds. This led to repositioning of Niclosamide (Niclo) and Pyrvinium Pamoate (PP), two anthelmintic drugs, as inhibitors of oncogenic PI3K-dependent signalling. Niclo inhibited phosphorylation of P70S6K, while PP inhibited phosphorylation of AKT and P70S6K, which are downstream targets of PI3K. Anthelmintics inhibited oncogenic PI3K-dependent gene expression and showed a cytostatic effect in vitro and in mouse mammary gland. Lastly, PP inhibited the growth of breast cancer cells harbouring PI3K mutations. Our data indicate that drug repositioning by network analysis of oncogene-specific transcriptional signatures is an efficient strategy for identifying oncogenic pathway inhibitors among FDA-approved compounds. We propose that PP and Niclo should be further investigated as potential therapeutics for the treatment of tumors or diseases carrying the constitutive activation of the PI3K/P70S6K signalling axis.
- Published
- 2016
40. The genetic basis of undiagnosed muscular dystrophies and myopathies: Results from 504 patients
- Author
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Francesca Magri, Annalaura Torella, Corrado Angelini, Vincenzo Nigro, Luisa Politano, Olimpia Farina, Kathleen Claes, Roberta Petillo, Paola D'Ambrosio, Gabriele Siciliano, Enrico Bertini, Marina Fanin, Francesca Gualandi, Sonia Messina, Giorgio Tasca, Peter Hackman, Giulio Piluso, Alessandra Ruggieri, Simone Sanpaolo, Enzo Ricci, Jan De Bleecker, Lucia Ruggiero, Giacomo P. Comi, Sabrina Sacconi, Dario Ronchi, Adele D'Amico, Giuseppina Di Fruscio, Giulia Ricci, Eugenio Mercuri, Giuseppe Di Iorio, Chiara Fiorillo, Maurizio Moggio, Liliana Vercelli, Tiziana Mongini, Claudio Bruno, Lorenzo Maggi, Olimpia Musumeci, Marina Mora, Veer Singh Marwah, Carlo Minetti, Carmelo Rodolico, L. Passamano, Bjarne Udd, Guja Astrea, Arcomaria Garofalo, Elena Pegoraro, Margherita Mutarelli, Gaia Esposito, Sandra Janssens, Anni Evilä, Massimiliano Filosto, Francesca Del Vecchio Blanco, Lucio Santoro, Antonio Toscano, Rossella Tupler, Marco Savarese, Teresa Giugliano, Filippo M. Santorelli, Savarese, M, Di Fruscio, G, Torella, Annalaura, Fiorillo, C, Magri, F, Fanin, M, Ruggiero, L, Ricci, G, Astrea, G, Passamano, L, Ruggieri, A, Ronchi, D, Tasca, G, D'Amico, A, Janssens, S, Farina, O, Mutarelli, M, Marwah, V, Garofalo, A, Giugliano, T, Sampaolo, Simone, DEL VECCHIO BLANCO, Francesca, Esposito, G, Piluso, Giulio, D'Ambrosio, P, Petillo, R, Musumeci, O, Rodolico, C, Messina, S, Evilä, A, Hackman, P, Filosto, M, DI IORIO, Giuseppe, Siciliano, G, Mora, M, Maggi, L, Minetti, C, Sacconi, S, Santoro, Laura, Claes, K, Vercelli, L, Mongini, T, Ricci, E, Gualandi, F, Tupler, R, De Bleecker, J, Udd, B, Toscano, A, Moggio, M, Pegoraro, E, Bertini, E, Mercuri, E, Angelini, C, Santorelli, Fm, Politano, Luisa, Bruno, C, Comi, Gp, and Nigro, Vincenzo
- Subjects
Male ,0301 basic medicine ,Pediatrics ,Pathology ,MENDELIAN DISEASE ,Eleventh ,Bioinformatics ,Muscular Dystrophies ,Cohort Studies ,0302 clinical medicine ,congenital myopathy ,030212 general & internal medicine ,Muscular dystrophy ,limb-girdle muscular dystrophy ,Phenotype ,MENDELIAN DISEASE, NEUROMUSCULAR DISORDERS, DIAGNOSIS, PHENOTYPES ,DUCHENNE ,3. Good health ,Italy ,Female ,medicine.symptom ,Sequence Analysis ,muscular dystrophy ,medicine.medical_specialty ,PHENOTYPES ,DIAGNOSIS ,Article ,Diagnosis, Differential ,03 medical and health sciences ,NEUROMUSCULAR DISORDERS ,Genetic variation ,medicine ,Humans ,Myopathy ,business.industry ,Genetic Variation ,Correction ,Regret ,Molecular diagnostics ,medicine.disease ,Congenital myopathy ,neuromuscular disorder ,030104 developmental biology ,Disease Presentation ,next-generation sequencing ,Neurology (clinical) ,Differential diagnosis ,business ,030217 neurology & neurosurgery - Abstract
OBJECTIVE: To apply next-generation sequencing (NGS) for the investigation of the genetic basis of undiagnosed muscular dystrophies and myopathies in a very large cohort of patients. METHODS: We applied an NGS-based platform named MotorPlex to our diagnostic workflow to test muscle disease genes with a high sensitivity and specificity for small DNA variants. We analyzed 504 undiagnosed patients mostly referred as being affected by limb-girdle muscular dystrophy or congenital myopathy. RESULTS: MotorPlex provided a complete molecular diagnosis in 218 cases (43.3%). A further 160 patients (31.7%) showed as yet unproven candidate variants. Pathogenic variants were found in 47 of 93 genes, and in more than 30% of cases, the phenotype was nonconventional, broadening the spectrum of disease presentation in at least 10 genes. CONCLUSIONS: Our large DNA study of patients with undiagnosed myopathy is an example of the ongoing revolution in molecular diagnostics, highlighting the advantages in using NGS as a first-tier approach for heterogeneous genetic conditions. Objective: To apply next-generation sequencing (NGS) for the investigation of the genetic basis of undiagnosed muscular dystrophies and myopathies in a very large cohort of patients. Methods: We applied an NGS-based platform namedMotorPlex to our diagnostic workflow to test muscle disease genes with a high sensitivity and specificity for small DNA variants. We analyzed 504 undiagnosed patients mostly referred as being affected by limb-girdle muscular dystrophy or congenital myopathy. Results: MotorPlex provided a complete molecular diagnosis in 218 cases (43.3%). A further 160 patients (31.7%) showed as yet unproven candidate variants. Pathogenic variants were found in 47 of 93 genes, and in more than 30%of cases, the phenotype was nonconventional, broadening the spectrum of disease presentation in at least 10 genes. Conclusions: Our large DNA study of patients with undiagnosed myopathy is an example of the ongoing revolution in molecular diagnostics, highlighting the advantages in using NGS as a first-tier approach for heterogeneous genetic conditions.
- Published
- 2016
41. DUX4 signature in STIR+ Facioscapulohumeral muscular dystrophy muscles
- Author
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A. Carissimo, Vincenzo Nigro, Margherita Mutarelli, Arcomaria Garofalo, Mauro Monforte, Enzo Ricci, Giorgio Tasca, and Mario Pescatori
- Subjects
Pathology ,medicine.medical_specialty ,Neurology ,DUX4 ,business.industry ,Pediatrics, Perinatology and Child Health ,medicine ,Facioscapulohumeral muscular dystrophy ,Neurology (clinical) ,medicine.disease ,Signature (topology) ,business ,Genetics (clinical) - Published
- 2017
42. HOCTAR database: A unique resource for microRNA target prediction
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Gopuraja Dharmalingam, Giampiero Lago, Vincenza Maselli, Margherita Mutarelli, Vincenzo A. Gennarino, Marco Sardiello, Sandro Banfi, Gennarino, Va, Sardiello, M, Mutarelli, M, Dharmalingam, G, Maselli, V, Lago, G, and Banfi, Sandro
- Subjects
Databases, Factual ,Translational Inhibition ,Biology ,computer.software_genre ,Article ,Mirna target ,Transcriptome ,AKT1, v-akt murine thymoma viral oncogene homolog 1 ,Target prediction ,microRNA ,Genetics ,Humans ,TFEB, transcription factor EB ,Gene ,Regulation of gene expression ,Database ,Gene ontology ,MRNA cleavage ,Computational Biology ,Reproducibility of Results ,General Medicine ,MicroRNAs ,Gene Expression Regulation ,HTT, huntingtin ,TMEM49, transmembrane protein 49 ,computer - Abstract
microRNAs (miRNAs) are the most abundant class of small RNAs in mammals. They play an important role in regulation of gene expression by inducing mRNA cleavage or translational inhibition. Each miRNA targets an average of 100–200 genes by binding, preferentially, to their 3′ UTRs by means of partial sequence complementarity. Most miRNAs are localized within transcriptional units, termed host genes, and show similar expression behavior with respect to their corresponding host genes. Considering the impact of miRNA in the regulation of gene expression and their involvement in a growing number of human disorders, it is vital to develop sensitive computational approaches able to identify miRNA target genes. The HOCTAR database (db) is a publicly available resource collecting ranked list of predicted target genes for 290 intragenic miRNAs annotated in human. HOCTARdb is a unique resource that integrates miRNA target prediction genes and transcriptomic data to score putative miRNA targets looking at the expression behavior of their host genes. We demonstrated, by testing 135 known validated target genes (either at the translational or transcriptional level) for different miRNAs, that the miRNA target prediction lists present in HOCTARdb are highly reliable. Moreover, HOCTARdb associates biological roles to each miRNA-controlled transcriptional network by means of Gene Ontology analysis. This information is easily accessible through a user-friendly query page. The HOCTARdb is available at http://hoctar.tigem.it/. We believe that a detailed relationship between miRNAs and their target genes and a constant update of the information contained in HOCTARdb will provide an extremely valuable resource to assist the researcher in the discovery of miRNA target genes.
- Published
- 2011
43. Quantitative expression profiling of highly degraded RNA from formalin-fixed, paraffin-embedded breast tumor biopsies by oligonucleotide microarrays
- Author
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Alessandra Vigilante, Margherita Mutarelli, Ornella Paris, Maria Ravo, Olì M. V. Grober, Alessandro Weisz, Roberta Tarallo, E. Nola, Luigi Cicatiello, Daniela Cimino, Lorenzo Ferraro, Michele De Bortoli, Ravo, M., Mutarelli, M., Ferraro, L., Grober, O. M. V., Paris, O., Tarallo, R., Vigilante, A., Cimino, D., DE BORTOLI, M., Nola, Ernesto, Cicatiello, L., and Weisz, A.
- Subjects
Microarray ,breast cancer ,formalin-fixed tissues ,microarrays ,Gene Expression ,Biopsy ,Breast Neoplasms ,Biology ,Pathology and Forensic Medicine ,Cell Line, Tumor ,Formaldehyde ,Gene expression ,medicine ,Humans ,RNA, Neoplasm ,Molecular Biology ,Oligonucleotide Array Sequence Analysis ,Paraffin Embedding ,medicine.diagnostic_test ,Microarray analysis techniques ,Gene Expression Profiling ,Carcinoma, Ductal, Breast ,Reproducibility of Results ,RNA ,Cell Biology ,Molecular biology ,In vitro ,Gene expression profiling ,expression profiling ,Female ,DNA microarray ,microarray - Abstract
Microarray-based gene expression profiling is well suited for parallel quantitative analysis of large numbers of RNAs, but its application to cancer biopsies, formalin-fixed paraffin-embedded (FFPE) archived tissues in particular, is limited by the poor quality of the RNA recovered. This represents a serious drawback, since FFPE tumor tissue banks are available with clinical and prognostic annotations, which could be exploited for molecular profiling studies, provided that reliable analytical technologies are found. We applied and evaluated here a microarray-based cDNA-mediated annealing, selection, extension and ligation (DASL) assay for analysis of 502 mRNAs in highly degraded total RNA extracted from cultured cells or FFPE breast cancer biopsies. The study included quantitative and qualitative comparison of data obtained by analysis of the same RNAs with genome-wide oligonucleotide microarrays vs. DASL arrays and, by DASL, before and after extensive in vitro RNA fragmentation. The DASL-based expression profiling assay applied to RNA extracted from MCF-7 cells before or after 24 hours stimulation with a mitogenic dose of 17β-estradiol consistently allowed to detect hormone-induced gene expression changes also following extensive RNA degradation in vitro. Comparable results where obtained with tumor RNA extracted from FFPE breast cancer biopsies (6 to 19 years old). The method proved itself sensitive, reproducible and accurate, when compared with results obtained by microarray analysis of RNA extracted from snap-frozen tissue of the same tumor.
- Published
- 2008
44. Are all the previously reported genetic variants in limb girdle muscular dystrophy genes pathogenic?
- Author
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Giuseppina Di Fruscio, Arcomaria Garofalo, Marco Savarese, Margherita Mutarelli, Vincenzo Nigro, Di Fruscio, G., Garofalo, A., Mutarelli, M., Savarese, M., and Nigro, Vincenzo
- Subjects
0301 basic medicine ,Male ,Genes, Recessive ,Nerve Tissue Proteins ,Penetrance ,Gene mutation ,Biology ,Article ,03 medical and health sciences ,Gene Frequency ,Nuclear Matrix-Associated Proteins ,Databases, Genetic ,Genetics ,medicine ,Humans ,Exome ,Allele ,Age of Onset ,Genetics (clinical) ,Exome sequencing ,Alleles ,Genes, Dominant ,Genetic Variation ,medicine.disease ,Cytoskeletal Proteins ,030104 developmental biology ,Muscular Dystrophies, Limb-Girdle ,Dystrophin-Associated Proteins ,Mutation ,Female ,Leiden Open Variation Database ,Limb-girdle muscular dystrophy ,Common disease-common variant - Abstract
Hundreds of variants in autosomal genes associated with the limb girdle muscular dystrophies (LGMDs) have been reported as being causative. However, in most cases the proof of pathogenicity derives from their non-occurrence in hundreds of healthy controls and/or from segregation studies in small families. The limited statistics of the genetic variations in the general population may hamper a correct interpretation of the effect of variants on the protein. To clarify the meaning of low-frequency variants in LGMD genes, we have selected all variants described as causative in the Leiden Open Variation Database and the Human Gene Mutation Database. We have systematically searched for their frequency in the NHLBI GO Exome Sequencing Project (ESP) and in our internal database. Surprisingly, the ESP contains about 4% of the variants previously associated with a dominant inheritance and about 9% of those associated with a recessive inheritance. The putative disease alleles are much more frequent than those estimated considering the disease prevalence. In conclusion, we hypothesize that a number of disease-associated variants are non-pathogenic and that other variations are not fully penetrant, even if they affect the protein function, suggesting a more complex genetic mechanisms for such heterogeneous disorders.
- Published
- 2015
45. MotorPlex provides accurate variant detection across large muscle genes both in single myopathic patients and in pools of DNA samples
- Author
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Claudio Bruno, Annalaura Torella, Filippo M. Santorelli, Giuseppina Di Fruscio, Margherita Mutarelli, Marco Savarese, Giacomo P. Comi, Francesca Magri, and Vincenzo Nigro
- Subjects
Pooling ,Male ,Nonsynonymous substitution ,Candidate gene ,Muscular dystrophies ,Population ,Muscle disorder ,Sensitivity and Specificity ,DNA sequencing ,Pathology and Forensic Medicine ,Cellular and Molecular Neuroscience ,Muscular Diseases ,Next generation sequencing ,Genetic variation ,Humans ,Medicine ,Computer Simulation ,Muscle, Skeletal ,education ,Exome sequencing ,Genetics ,education.field_of_study ,business.industry ,Research ,Genetic Variation ,Sequence Analysis, DNA ,Myopathies ,Female ,Human genome ,Neurology (clinical) ,business ,Target sequencing - Abstract
Mutations in ~100 genes cause muscle diseases with complex and often unexplained genotype/phenotype correlations. Next-generation sequencing studies identify a greater-than-expected number of genetic variations in the human genome. This suggests that existing clinical monogenic testing systematically miss very relevant information. We have created a core panel of genes that cause all known forms of nonsyndromic muscle disorders (MotorPlex). It comprises 93 loci, among which are the largest and most complex human genes, such as TTN, RYR1, NEB and DMD. MotorPlex captures at least 99.2% of 2,544 exons with a very accurate and uniform coverage. This quality is highlighted by the discovery of 20-30% more variations in comparison with whole exome sequencing. The coverage homogeneity has also made feasible to apply a cost-effective pooled sequencing strategy while maintaining optimal sensitivity and specificity. We studied 177 unresolved cases of myopathies for which the best candidate genes were previously excluded. We have identified known pathogenic variants in 52 patients and potential causative ones in further 56 patients. We have also discovered 23 patients showing multiple true disease-associated variants suggesting complex inheritance. Moreover, we frequently detected other nonsynonymous variants of unknown significance in the largest muscle genes. Cost-effective combinatorial pools of DNA samples were similarly accurate (97-99%). MotorPlex is a very robust platform that overcomes for power, costs, speed, sensitivity and specificity the gene-by-gene strategy. The applicability of pooling makes this tool affordable for the screening of genetic variability of muscle genes also in a larger population. We consider that our strategy can have much broader applications. Electronic supplementary material The online version of this article (doi:10.1186/s40478-014-0100-3) contains supplementary material, which is available to authorized users.
- Published
- 2014
46. A community-based resource for automatic exome variant-calling and annotation in Mendelian disorders
- Author
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Gopuraja Dharmalingam, Diego Carrella, Veer Singh Marwah, Margherita Mutarelli, Gennaro Oliva, Diego di Bernardo, Rossella Rispoli, M., Mutarelli, V., Marwah, R., Rispoli, D., Carrella, G., Dharmalingam, G., Oliva, and DI BERNARDO, Diego
- Subjects
Population ,Biology ,Web Browser ,Polymorphism, Single Nucleotide ,Workflow ,whole exome sequencing ,symbols.namesake ,INDEL Mutation ,Databases, Genetic ,Genetics ,Humans ,Exome ,education ,Allele frequency ,mendelian disorders ,Exome sequencing ,education.field_of_study ,Research ,Genetic Diseases, Inborn ,Computational Biology ,Genetic Variation ,High-Throughput Nucleotide Sequencing ,Molecular Sequence Annotation ,Pipeline (software) ,high performance computing ,variant database ,Mendelian inheritance ,symbols ,Database Management Systems ,Software ,Reference genome ,Biotechnology - Abstract
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. 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. 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.
- Published
- 2014
47. MIB2variants altering NOTCH signalling result in left ventricle hypertrabeculation/non-compaction and are associated with Ménétrier-like gastropathy
- Author
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Mauro Giacca, Annamaria Staiano, Pasquale Piccolo, Ty C. Lynnes, Caterina Strisciuglio, Alfonso Valencia, Katherine G. Spoonamore, Matteo Vatta, Margherita Mutarelli, Chiara Collesi, Francesco Paolo D'Armiento, Erasmo Miele, Severo Campione, Ilaria Secco, Giuseppe Limongelli, Lorena Zentilin, Nicola Brunetti-Pierri, Tirso Pons, Gerardo Nardone, Patrícia B. S. Celestino-Soper, Sergio Attanasio, Sandro Banfi, Vincenzo Nigro, Riccardo Sangermano, Piccolo, Pasquale, Attanasio, Sergio, Secco, Ilaria, Sangermano, Riccardo, Strisciuglio, Caterina, Limongelli, Giuseppe, Miele, Erasmo, Mutarelli, Margherita, Banfi, Sandro, Nigro, Vincenzo, Pons, Tirso, Valencia, Alfonso, Zentilin, Lorena, Campione, Severo, Nardone, Gerardo, Lynnes, Ty C, Celestino-Soper, Patricia B S, Spoonamore, Katherine G, D'Armiento, Francesco P, Giacca, Mauro, Staiano, Annamaria, Vatta, Matteo, Collesi, Chiara, Brunetti-Pierri, Nicola, Lynnes, Ty C., Celestino Soper, Patricia B. S., Spoonamore, Katherine G., D'Armiento, Francesco P., Brunetti Pierri, Nicola, and Celestino Soper, Patricia B. S
- Subjects
Male ,0301 basic medicine ,Heart malformation ,Ubiquitin-Protein Ligases ,Mutation, Missense ,Stomach Diseases ,Notch signaling pathway ,Biology ,medicine.disease_cause ,Ventricular Dysfunction, Left ,03 medical and health sciences ,0302 clinical medicine ,Genetic ,Molecular Biology ,Genetics ,Genetics (clinical) ,medicine ,Animals ,Humans ,Missense mutation ,Exome ,Myocytes, Cardiac ,HES1 ,Gastritis, Hypertrophic ,Cells, Cultured ,Exome sequencing ,Mutation ,Receptors, Notch ,Ubiquitination ,General Medicine ,Pedigree ,Rats ,Phenotype ,030104 developmental biology ,Animals, Newborn ,Gene Expression Regulation ,Case-Control Studies ,Cancer research ,Left ventricular noncompaction ,Female ,Cardiomyopathies ,030217 neurology & neurosurgery ,Signal Transduction - Abstract
We performed whole exome sequencing in individuals from a family with autosomal dominant gastropathy resembling Méné trier disease, a premalignant gastric disorder with epithelial hyperplasia and enhanced EGFR signalling. Méné trier disease is believed to be an acquired disorder, but its aetiology is unknown. In affected members, we found a missense p.V742G variant in MIB2, a gene regulating NOTCH signalling that has not been previously linked to human diseases. The variant segregated with the disease in the pedigree, affected a highly conserved amino acid residue, and was predicted to be deleterious although it was found with a low frequency in control individuals. The purified protein carrying the p.V742G variant showed reduced ubiquitination activity in vitro and white blood cells from affected individuals exhibited significant reductions of HES1 and NOTCH3 expression reflecting alteration of NOTCH signalling. Because mutations of MIB1, the homolog of MIB2, have been found in patients with left ventricle non-compaction (LVNC), we investigated members of our family with Méné trier-like disease for this cardiac abnormality. Asymptomatic left ventricular hypertrabeculation, the mildest end of the LVNC spectrum, was detected in two members carrying the MIB2 variant. Finally, we identified an additional MIB2 variant (p.V984L) affecting protein stability in an unrelated isolated case with LVNC. Expression of both MIB2 variants affected NOTCH signalling, proliferation and apoptosis in primary rat cardiomyocytes. In conclusion, we report the first example of left ventricular hypertrabeculation/LVNC with germline MIB2 variants resulting in altered NOTCH signalling that might be associated with a gastropathy clinically overlapping with Méné trier disease.
- Published
- 2016
48. Next-generation sequencing identifies transportin 3 as the causative gene for LGMD1F
- Author
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Marco Savarese, Lucia Morandi, Giulia Ricci, Giulio Piluso, Francesca Del Vecchio Blanco, Gabriele Siciliano, Marina Fanin, Annalaura Torella, Margherita Mutarelli, Corrado Angelini, Rossella Rispoli, Arcomaria Garofalo, Vincenzo Nigro, Enrico Peterle, Torella, A, Fanin, M, Mutarelli, M, Peterle, E, Del Vecchio Blanco, F, Rispoli, R, Savarese, M, Garofalo, A, Piluso, Giulio, Morandi, L, Ricci, G, Siciliano, G, Angelini, C, and Nigro, Vincenzo
- Subjects
Proteomics ,Male ,Mutant ,Gene Expression ,Fluorescent Antibody Technique ,lcsh:Medicine ,Protein Synthesis ,medicine.disease_cause ,Biochemistry ,Muscular Dystrophies ,Molecular Cell Biology ,Sequencing ,Missense mutation ,Exome ,Genome Sequencing ,Muscular dystrophy ,lcsh:Science ,Genetics ,Mutation ,Multidisciplinary ,Genomics ,beta Karyopherins ,Pedigree ,Neurology ,Medicine ,Female ,Sequence Analysis ,Research Article ,Immunology ,Blotting, Western ,Molecular Sequence Data ,Biology ,Gene product ,Genome Analysis Tools ,medicine ,Humans ,Genetic Predisposition to Disease ,Genetic Testing ,Immunoassays ,Muscle, Skeletal ,Gene ,Clinical Genetics ,Sequence Assembly Tools ,Base Sequence ,Point mutation ,lcsh:R ,Proteins ,Computational Biology ,Sequence Analysis, DNA ,medicine.disease ,Muscular Dystrophies, Limb-Girdle ,Immunologic Techniques ,Protein Translation ,Mutant Proteins ,lcsh:Q ,HeLa Cells - Abstract
"Limb-girdle muscular dystrophies (LGMD) are genetically and clinically heterogeneous conditions. We investigated a large family with autosomal dominant transmission pattern, previously classified as LGMD1F and mapped to chromosome 7q32. Affected members are characterized by muscle weakness affecting earlier the pelvic girdle and the ileopsoas muscles. We sequenced the whole exome of four family members and identified a shared heterozygous frame-shift variant in the Transportin 3 (TNPO3) gene, encoding a member of the importin-β super-family. The TNPO3 gene is mapped within the LGMD1F critical interval and its 923-amino acid human gene product is also expressed in skeletal muscle. In addition, we identified an isolated case of LGMD with a new missense mutation in the same gene. We localized the mutant TNPO3 around the nucleus, but not inside. The involvement of gene related to the nuclear transport suggests a novel disease mechanism leading to muscular dystrophy."
- Published
- 2013
49. Molecular diagnosis of usher syndrome: application of two different next generation sequencing-based procedures
- Author
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Emmanouil Athanasakis, Ivana Peluso, Angela D'Eustacchio, Vincenzo Nigro, Antonella Fabretto, Carmela Ziviello, Nienke Wieskamp, Danilo Licastro, Margherita Mutarelli, Francesca Simonelli, Paolo Gasparini, Rossella Rispoli, F. d’Amico, Kornelia Neveling, Diego Vozzi, Mariateresa Pizzo, Hans Scheffer, Sandro Banfi, Licastro, Danilo, Mutarelli, Margherita, Peluso, Ivana, Neveling, Kornelia, Wieskamp, Nienke, Rispoli, Rossella, Vozzi, Diego, Athanasakis, Emmanouil, D'Eustacchio, Angela, Pizzo, Mariateresa, D'Amico, Francesca, Ziviello, Carmela, Simonelli, Francesca, Fabretto, Antonella, Scheffer, Han, Gasparini, Paolo, Banfi, Sandro, Nigro, Vincenzo, Licastro, D, Mutarelli, M, Peluso, I, Neveling, K, Wieskamp, N, Rispoli, R, Vozzi, D, Athanasakis, E, D'Eustacchio, A, Pizzo, M, D'Amico, F, Ziviello, C, Fabretto, A, Scheffer, H, and Gasparini, P
- Subjects
Genetics and Molecular Biology (all) ,Genetic Screens ,Gene Identification and Analysis ,lcsh:Medicine ,Pilot Projects ,Biochemistry ,Genome Databases ,Exome ,Genome Sequencing ,lcsh:Science ,Child ,Exome sequencing ,Genetics ,0303 health sciences ,Multidisciplinary ,Massive parallel sequencing ,Genome ,Medicine (all) ,030305 genetics & heredity ,High-Throughput Nucleotide Sequencing ,Genomics ,3. Good health ,Molecular Diagnostic Techniques ,Child, Preschool ,Medicine ,Usher Syndrome ,Genome, Human ,Humans ,Sequence Analysis, DNA ,Usher Syndromes ,Agricultural and Biological Sciences (all) ,Biochemistry, Genetics and Molecular Biology (all) ,Sequence Analysis ,Research Article ,Human ,Molecular Diagnostic Technique ,Sequence Databases ,Genetic Counseling ,Biology ,DNA sequencing ,Genomic disorders and inherited multi-system disorders DCN MP - Plasticity and memory [IGMD 3] ,Genomic disorders and inherited multi-system disorders [IGMD 3] ,Molecular Genetics ,03 medical and health sciences ,Pilot Project ,Genetic Testing ,Preschool ,Genotyping ,030304 developmental biology ,Clinical Genetics ,lcsh:R ,Personalized Medicine ,Human Genetics ,DNA ,Molecular diagnostics ,Otorhinolaryngology ,Genetics of Disease ,Mutation Databases ,Human genome ,lcsh:Q - Abstract
Contains fulltext : 108716.pdf (Publisher’s version ) (Open Access) Usher syndrome (USH) is a clinically and genetically heterogeneous disorder characterized by visual and hearing impairments. Clinically, it is subdivided into three subclasses with nine genes identified so far. In the present study, we investigated whether the currently available Next Generation Sequencing (NGS) technologies are already suitable for molecular diagnostics of USH. We analyzed a total of 12 patients, most of which were negative for previously described mutations in known USH genes upon primer extension-based microarray genotyping. We enriched the NGS template either by whole exome capture or by Long-PCR of the known USH genes. The main NGS sequencing platforms were used: SOLiD for whole exome sequencing, Illumina (Genome Analyzer II) and Roche 454 (GS FLX) for the Long-PCR sequencing. Long-PCR targeting was more efficient with up to 94% of USH gene regions displaying an overall coverage higher than 25x, whereas whole exome sequencing yielded a similar coverage for only 50% of those regions. Overall this integrated analysis led to the identification of 11 novel sequence variations in USH genes (2 homozygous and 9 heterozygous) out of 18 detected. However, at least two cases were not genetically solved. Our result highlights the current limitations in the diagnostic use of NGS for USH patients. The limit for whole exome sequencing is linked to the need of a strong coverage and to the correct interpretation of sequence variations with a non obvious, pathogenic role, whereas the targeted approach suffers from the high genetic heterogeneity of USH that may be also caused by the presence of additional causative genes yet to be identified.
- Published
- 2012
50. Global analysis of estrogen receptor beta binding to breast cancer cell genome reveals an extensive interplay with estrogen receptor alpha for target gene regulation
- Author
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Maria Francesca Papa, Maria R. De Filippo, Vladimir Benes, Shujun Luo, Olì M. V. Grober, Margherita Mutarelli, Alessandro Weisz, Luigi Cicatiello, Maria Ravo, Giorgio Giurato, Roberta Tarallo, Ornella Paris, Gary P. Schroth, Lorenzo Ferraro, and Giovanni Nassa
- Subjects
Chromatin Immunoprecipitation ,lcsh:QH426-470 ,lcsh:Biotechnology ,Immunoblotting ,Estrogen receptor ,Biology ,Cell Line, Tumor ,lcsh:TP248.13-248.65 ,Genetics ,Estrogen Receptor beta ,Humans ,skin and connective tissue diseases ,E2F ,Transcription factor ,Estrogen receptor beta ,Cell Proliferation ,Oligonucleotide Array Sequence Analysis ,Hormone response element ,Binding Sites ,Estrogen Receptor alpha ,Molecular biology ,Cell biology ,body regions ,Gene Expression Regulation, Neoplastic ,lcsh:Genetics ,Signal transduction ,Chromatin immunoprecipitation ,Estrogen receptor alpha ,Protein Binding ,Research Article ,Biotechnology - Abstract
Background Estrogen receptors alpha (ERα) and beta (ERβ) are transcription factors (TFs) that mediate estrogen signaling and define the hormone-responsive phenotype of breast cancer (BC). The two receptors can be found co-expressed and play specific, often opposite, roles, with ERβ being able to modulate the effects of ERα on gene transcription and cell proliferation. ERβ is frequently lost in BC, where its presence generally correlates with a better prognosis of the disease. The identification of the genomic targets of ERβ in hormone-responsive BC cells is thus a critical step to elucidate the roles of this receptor in estrogen signaling and tumor cell biology. Results Expression of full-length ERβ in hormone-responsive, ERα-positive MCF-7 cells resulted in a marked reduction in cell proliferation in response to estrogen and marked effects on the cell transcriptome. By ChIP-Seq we identified 9702 ERβ and 6024 ERα binding sites in estrogen-stimulated cells, comprising sites occupied by either ERβ, ERα or both ER subtypes. A search for TF binding matrices revealed that the majority of the binding sites identified comprise one or more Estrogen Response Element and the remaining show binding matrixes for other TFs known to mediate ER interaction with chromatin by tethering, including AP2, E2F and SP1. Of 921 genes differentially regulated by estrogen in ERβ+ vs ERβ- cells, 424 showed one or more ERβ site within 10 kb. These putative primary ERβ target genes control cell proliferation, death, differentiation, motility and adhesion, signal transduction and transcription, key cellular processes that might explain the biological and clinical phenotype of tumors expressing this ER subtype. ERβ binding in close proximity of several miRNA genes and in the mitochondrial genome, suggests the possible involvement of this receptor in small non-coding RNA biogenesis and mitochondrial genome functions. Conclusions Results indicate that the vast majority of the genomic targets of ERβ can bind also ERα, suggesting that the overall action of ERβ on the genome of hormone-responsive BC cells depends mainly on the relative concentration of both ERs in the cell.
- Published
- 2011
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