Genomic and Genetic Disorders Biobank

The Genomic and Genetic Disorders Biobank (GGDB, formerly Genomic Disorders Biobank) was established in 2006 as an internal bioresource supported by own database, to collect and store biospecimens from genomic-disorder (e.g. microdeletion and duplication syndromes) affected individuals. Since 2009 the Bioresource has joined the Telethon Network of Genetic Biobanks and has expanded the collection to include also genetic mendelian diseases. The GGDB gathers wholly annotated clinical and longitudinal data and biological samples from affected and healthy donors, according to standard ethical principles. Biospecimens are available to the international scientific community for research projects in the field of the pathologies collected and stored in GGDB.</p

MODULAZIONE DELL'ATTIVAZIONE DEI MONOCITI DURANTE L'INFIAMMAZIONE: ANALISI INTEGRATA DI DATI TRASCRITTOMICI E PROTEOMICI DELLA FAMIGLIA DI CITOCHINE IL-1/IL-18

L’infiammazione è una risposta di difesa che viene innescata da stimoli e condizioni nocive, quali una infezione o un danno tissutale. Nel sito dove inizia l’evento infiammatorio, le cellule esposte all’insulto producono una serie di citochine e chemiochine che agiscono sull’endotelio vascolare locale, causando dilatazione dei vasi sanguigni, fuoriuscita di liquidi e reclutamento di neutrofili e monociti dal sangue nel tessuto. Brevemente, l’iniziale riconoscimento di una infezione o l’intervento su un danno tissutale avvengono da parte, fra le altre cellule, dei macrofagi residenti, che in risposta allo stimolo producono una varietà di mediatori della infiammazione, incluse chemiochine (es. MCP-1), citochine (es. TNF-α e IL-1β), amine vasoattive, prostaglandine. In risposta a tali fattori si forma l’essudato infiammatorio locale: proteine plasmatiche e leucociti (neutrofili e monociti), escono dal circolo e accedono al tessuto nel sito di infezione/danno. Una volta nel tessuto danneggiato, monociti e neutrofili si attivano (o attraverso il contatto diretto con l’agente patogeno o attraverso l’azione di citochine secrete dalle cellule residenti nel tessuto) e, nel tentativo di eliminare l’agente invasore, rilasciano fattori tossici (specie reattive dell’ossigeno e dell’azoto, proteasi, elastasi, collagenasi). Tali fattori, non discriminando tra bersagli microbici e tessuti dell’ospite, provocano danni ai tessuti come effetto collaterale dell’attività di difesa. Ne segue che, pur essendo un evento essenziale per la difesa dell’integrità dell’organismo dagli attacchi esterni, la risposta infiammatoria necessita di uno stretto controllo della propria attivazione ma soprattutto che abbia termine una volta eliminato l’agente pericoloso che l’ha scatenata in modo da non causare danni rilevanti all’organismo stesso. Nel corso dello sviluppo e della risoluzione dell’infiammazione sono di particolare importanza i fagociti mononucleati (sia i monociti circolanti, cellule meno mature che entrano nel tessuto infiammato in risposta a chemiochine, che i macrofagi residenti nel tessuto, cellule mature che sorvegliano e controllano l’integrità tissutale). I fagociti mononucleati sono cellule del sistema immunitario innato essenziali per la difesa dell’ospite, non solo perché effettori dell’eliminazione diretta degli agenti esterni (es. tramite fagocitosi o uccisione dei microbi), ma anche perché organizzatori delle fasi di inizio e di risoluzione dell’infiammazione. I macrofagi sono infatti cellule altamente plastiche, che possono rispondere a cambiamenti sottili del microambiente tissutale iniziando diversi programmi di attivazione. L’attivazione dei macrofagi avviene secondo due principali tipi di programma: l’attivazione infiammatoria classica (o M1), i cui stimoli attivanti prototipici sono molecole batteriche (es. LPS) e citochine infiammatorie (es. IFN-γ), e l’attivazione alternativa (o M2), i cui stimoli attivanti sono citochine anti-infiammatorie ( es. IL-4 e IL-10, TGF-β), immunocomplessi o glucocorticoidi. La risposta infiammatoria iniziale attiva la polarizzazione M1 dei macrofagi, che diventano capaci di eliminare i microorganismi invasori e promuovono la risposta infiammatoria, mentre durante la fase di risoluzione dell’infiammazione i macrofagi vengono ripolarizzati in direzione M2, perdendo la reattività agli stimoli infiammatori e assumendo la capacità di eliminare cellule e tessuti danneggiati, e di promuovere angiogenesi e rimodellamento tissutale. La mancata regolazione dei processi infiammatori è alla base di malattie infiammatorie croniche e autoimmuni, come l’artrite reumatoide, la sclerosi multipla e il lupus eritromatoso sistemico. La famiglia delle citochine IL-1 comprende proteine implicate nella risposta infiammatoria e anti-infiammatoria ed è noto che difetti nei meccanismi di controllo della loro produzione e/o dell’attività correlano con le suddette malattie. All’interno di questo contesto si colloca il seguente lavoro di tesi il cui scopo è stato quello di creare un sistema modello in vitro, basato su monociti umani del sangue che differenziano in macrofagi M1 ed M2, al fine di simulare i vari stadi del processo infiammatorio, dalla sua attivazione alla risoluzione. A diversi tempi durante la reazione infiammatoria vengono prelevati campioni di cellule e supernatanti per l’analisi dell’espressione genica (mediante real-time PCR) e della produzione proteica (mediante ELISA e Proteome Array), con particolare attenzione al comportamento dei membri della famiglia delle citochine IL-1 e loro recettori

Angiogenesis in NENs, with a focus on gastroenteropancreatic NENs: from biology to current and future therapeutic implications

Neuroendocrine neoplasms (NENs) are highly vascularized malignancies arising from cells of the diffuse neuroendocrine system. An intricated cross-talk exists between NEN cells and the tumor microenvironment, and three main molecular circuits (VEGF/VEGFR pathway, FGF-dependent signaling and PDGF/PDGFR axis) have been shown to regulate angiogenesis in these neoplasms. Multiple randomized trials have investigated antiangiogenic agents over the past two decades, and sunitinib is currently approved for the treatment of advanced, progressive, G1/G2 pancreatic NENs. In recent years, two phase III clinical trials have demonstrated the efficacy and safety of surufatinib, a multi-tyrosine kinase angioimmune inhibitor, in patients with well-differentiated pancreatic and extrapancreatic NENs, and two studies of this agent are currently underway in Europe and US. The HIF-2 alpha inhibitor belzutifan has recently received regulatory approval for the treatment of tumors arising in the context of Von-Hippel Lindau syndrome including pancreatic NENs, and a study of this drug in patients with sporadic tumors is presently ongoing. Combinations of antiangiogenic agents with chemotherapeutics and targeted drugs have been tested, with accumulating toxicities being a matter of concern. The potential of antiangiogenic agents in fine-tuning the immune microenvironment of NENs to enhance the activity of immune checkpoint inhibitors has been only partially elucidated, and further research should be carried out at this regard. Here, we review the current understanding of the biology of angiogenesis in NENs and provide a summary of the latest clinical investigations on antiangiogenic drugs in this malignancy

Cutting Edge: The NLRP3 Inflammasome Links Complement-Mediated Inflammation and IL-1β Release

The complement system is a potent component of the innate immune response, promoting inflammation and orchestrating defense against pathogens. However, dysregulation of complement is critical to several autoimmune and inflammatory syndromes. Elevated expression of the proinflammatory cytokine IL-1β is often linked to such diseases. In this study, we reveal the mechanistic link between complement and IL-1β secretion using murine dendritic cells. IL-1β secretion occurs following intracellular caspase-1 activation by inflammasomes. We show that complement elicits secretion of both IL-1β and IL-18 in vitro and in vivo via the NLRP3 inflammasome. This effect depends on the inflammasome components NLRP3 and ASC, as well as caspase-1 activity. Interestingly, sublethal complement membrane attack complex formation, but not the anaphylatoxins C3a and C5a, activated the NLRP3 inflammasome in vivo. These findings provide insight into the molecular processes underlying complement-mediated inflammation and highlight the possibility of targeting IL-1β to control complement-induced disease and pathological inflammation

gene2drug: a Computational Tool for Pathway-based Rational Drug Repositioning

Motivation Drug repositioning has been proposed as an effective shortcut to drug discovery. The availability of large collections of transcriptional responses to drugs enables computational approaches to drug repositioning directly based on measured molecular effects. Results We introduce a novel computational methodology for rational drug repositioning, which exploits the transcriptional responses following treatment with small molecule. Specifically, given a therapeutic target gene, a prioritization of potential effective drugs is obtained by assessing their impact on the transcription of genes in the pathway(s) including the target. We performed in silico validation and comparison with a state-of-art technique based on similar principles. We next performed experimental validation in two different real-case drug repositioning scenarios: (i) upregulation of the glutamate-pyruvate transaminase (GPT), which has been shown to induce reduction of oxalate levels in a mouse model of primary hyperoxaluria, and (ii) activation of the transcription factor TFEB, a master regulator of lysosomal biogenesis and autophagy, whose modulation may be beneficial in neurodegenerative disorders. Availability and implementation A web tool for Gene2drug is freely available at http://gene2drug.tigem.it. An R package is under development and can be obtained from https://github.com/franapoli/gep2pep

DAXX mutations as potential genomic markers of malignant evolution in small nonfunctioning pancreatic neuroendocrine tumors

Management of localized well-differentiated pancreatic neuroendocrine tumors (panNETs) is controversial and primarily dependent on tumor size. Upfront surgery is usually recommended for tumors larger than 2\u2009cm in diameter since they frequently show metastatic potential, whereas smaller panNETs are generally characterized by an indolent clinical course, with a rate of relapse or metastasis below 15%. To explore whether increased tumor size is paralleled by genomic variations, we compared the rate and the mutational patterns of putative driver genes that are recurrently altered in these tumors by investigating differential cohorts of panNET surgical specimens smaller (n\u2009=\u200927) or larger than 2\u2009cm (n\u2009=\u200929). We found that the cumulative number of mutations detected in panNETs &gt;2\u2009cm was significantly higher (p\u2009=\u20090.03) relative to smaller tumors, while mutations of DAXX were significantly more frequent in the cohort of larger tumors (p\u2009=\u20090.05). Moreover, mutations of DAXX were associated with features of malignancy including increased grade, nodal involvement and lymphovascular invasion, and independently predicted both relapse after surgery (p\u2009=\u20090.05) and reduced DFS in multivariable analysis (p\u2009=\u20090.02). Our data suggest that alterations of the DAXX/ATRX molecular machinery increase the malignant potential of panNETs, and that identification of mutations of DAXX/ATRX in small, nonfunctioning tumors can predict the malignant progression observed in a minority of them

Development of anti-somatostatin receptors CAR T cells for treatment of neuroendocrine tumors

Background Neuroendocrine tumors (NETs) overexpress somatostatin receptors (SSTRs). Methods We developed a second-generation, ligand-based, anti-SSTR chimeric antigen receptor (CAR) incorporating the somatostatin analog octreotide in its extracellular moiety. Results Anti-SSTR CAR T cells exerted antitumor activity against SSTR+NET cell linesin vitro. The killing activity was highly specific, as demonstrated by the lack of CAR T cell reactivity against NET cells engineered to express mutated variants of SSTR2/5 by CRISPR/Cas9. When adoptively transferred in NSG mice, anti-SSTR CAR T cells induced significant antitumor activity against human NET xenografts. Although anti-SSTR CAR T cells could recognize the murine SSTRs as shown by their killing ability against murine NET cells, no obvious deleterious effects on SSTR-expressing organs such as the brain or the pancreas were observed in mice. Conclusions Taken together, our results establish anti-SSTR CAR T cells as a potential candidate for early phase clinical investigations in patients with NETs. More broadly, the demonstration that a known peptide drug can direct CAR T cell targeting may streamline the potential utility of multiple peptide motifs and provide a blueprint for therapeutic applications in a variety of cancers

Identification of p53-target genes in Danio rerio

To orchestrate the genomic response to cellular stress signals, p53 recognizes and binds to DNA containing specific and well-characterized p53-responsive elements (REs). Differences in RE sequences can strongly affect the p53 transactivation capacity and occur even between closely related species. Therefore, the identification and characterization of a species-specific p53 Binding sistes (BS) consensus sequence and of the associated target genes may help to provide new insights into the evolution of the p53 regulatory networks across different species. Although p53 functions were studied in a wide range of species, little is known about the p53-mediated transcriptional signature in Danio rerio. Here, we designed and biochemically validated a computational approach to identify novel p53 target genes in Danio rerio genome. Screening all the Danio rerio genome by pattern-matching-based analysis, we found p53 RE-like patterns proximal to 979 annotated Danio rerio genes. Prioritization analysis identified a subset of 134 candidate pattern-related genes, 31 of which have been investigated in further biochemical assays. Our study identified runx1, axin1, traf4a, hspa8, col4a5, necab2, and dnajc9 genes as novel direct p53 targets and 12 additional p53-controlled genes in Danio rerio genome. The proposed combinatorial approach resulted to be highly sensitive and robust for identifying new p53 target genes also in additional animal species.This work was supported by Associazione Italiana per la Ricerca sul Cancro (AIRC, IG #14078), Ricerca Corrente 2014–16 granted by the Italian Ministry of Health, and the “5 × 1000” voluntary contributions to G.M., Ricerca Finalizzata 2011 granted by the Italian Ministry of Health to L.M. and partly supported by Ministero dell’Istruzione, dell’Università e della Ricerca (MIUR) ex-60% funds to M.M., E.M. end G.B.Peer reviewe

BRF1 mutations alter RNA polymerase III-dependent transcription and cause neurodevelopmental anomalies

RNA polymerase III (Pol III) synthesizes tRNAs and other small noncoding RNAs to regulate protein synthesis. Dysregulation of Pol III transcription has been linked to cancer, and germline mutations in genes encoding Pol III subunits or tRNA processing factors cause neurogenetic disorders in humans, such as hypomyelinating leukodystrophies and pontocerebellar hypoplasia. Here we describe an autosomal recessive disorder characterized by cerebellar hypoplasia and intellectual disability, as well as facial dysmorphic features, short stature, microcephaly, and dental anomalies. Whole-exome sequencing revealed biallelic missense alterations of BRF1 in three families. In support of the pathogenic potential of the discovered alleles, suppression or CRISPR-mediated deletion of bif1 in zebraflsh embryos recapitulated key neurodevelopmental phenotypes; in vivo complementation showed all four candidate mutations to be pathogenic in an apparent isoform-specific context. BRF1 associates with BDP1 and TBP to form the transcription factor IIIB (TFIIIB), which recruits Pol III to target genes. We show that disease-causing mutations reduce Brf1 occupancy at tRNA target genes in Saccharomyces cerevisiae and impair cell growth. Moreover, BRF1 mutations reduce Pol III-related transcription activity in vitro. Taken together, our data show that BRF1 mutations that reduce protein activity cause neurodevelopmental anomalies, suggesting that BRF1-mediated Pol III transcription is required for normal cerebellar and cognitive development