Literatura académica sobre el tema "Ngf phenotype"

Elevated bladder smooth muscle cell (BSMC) nerve growth factor (NGF) secretion and related neuroplasticity are associated with hyperactive voiding in spontaneously hypertensive rats (SHRs: hypertensive, behaviorally hyperactive), compared with control Wistar-Kyotos (WKYs). We used two inbred strains (WKHT: hypertensive; WKHA: hyperactive) to further investigate this phenomenon. WKHA BSMCs secreted higher basal levels of NGF than WKHT BSMCs. Antagonists did inhibit NGF output in WKHA but not WKHT cultures. Thus augmented basal secretion of NGF cosegregates with a hyperactive phenotype, whereas a lack of regulatory inhibition of NGF output cosegregates with a hypertensive phenotype. Bladder norepinephrine content paralleled NGF content, with WKHTs > SHRs > WKHAs > WKYs, providing evidence that a lack of inhibition is the greatest contributor to elevated bladder NGF and noradrenergic innervation. Protein kinase C (PKC) agonists affected NGF production differentially depending on strain, suggesting that altered PKC signaling may contribute to strain differences in NGF secretion. Finally, 6-h voiding frequency differed between the strains, with SHRs > WKHTs = WKHAs > WKYs. Thus aspects of both the hypertensive and hyperactive phenotypes may be associated with elevated SHR bladder NGF and hyperactive voiding.

The cause of the loss of basal forebrain cholinergic neurons (BFCNs) and their terminal synapses in the cerebral cortex and hippocampus in Alzheimer’s disease (AD) has provoked a decades-long controversy. The cholinergic phenotype of this neuronal system, involved in numerous cognitive mechanisms, is tightly dependent on the target-derived nerve growth factor (NGF). Consequently, the loss of BFCNs cholinergic phenotype in AD was initially suspected to be due to an NGF trophic failure. However, in AD there is a normal NGF synthesis and abundance of the NGF precursor (proNGF), therefore the NGF trophic failure hypothesis for the atrophy of BCNs was abandoned. In this review, we discuss the history of NGF-dependency of BFCNs and the atrophy of these neurons in Alzheimer’s disease (AD). Further to it, we propose that trophic factor failure explains the BFCNs atrophy in AD. We discuss evidence of the occurrence of a brain NGF metabolic pathway, the dysregulation of which, in AD explains the severe deficiency of NGF trophic support for the maintenance of BFCNs cholinergic phenotype. Finally, we revise recent evidence that the NGF metabolic dysregulation in AD pathology starts at preclinical stages. We also propose that the alteration of NGF metabolism-related markers in body fluids might assist in the AD preclinical diagnosis.

The PC12 pheochromocytoma cell line responds to NGF by undergoing growth arrest and proceeding to differentiate toward a neuronal phenotype. Among the early genetic events triggered by NGF in PC12 cells are the rapid activation of the zinc finger transcription factor Egr1/NGFI-A, and a slightly delayed induction of NAB2, a corepressor that inhibits Egr1 transcriptional activity. We found that stably transfected PC12 cells expressing high levels of NAB2 do not differentiate, but rather continue to proliferate in response to NGF. Inhibition of PC12 differentiation by NAB2 overexpression was confirmed using two additional experimental approaches, transient transfection, and adenoviral infection. Early events in the NGF signaling cascade, such as activation of MAP kinase and induction of immediate-early genes, were unaltered in the NAB2-overexpressing PC12 cell lines. However, induction of delayed NGF response genes such as TGF-β1 and MMP-3 was inhibited. Furthermore, NAB2 overexpression led to downregulation of p21WAF1, a molecule previously shown to play a pivotal role in the ability of PC12 cells to undergo growth arrest and commit to differentiation in response to NGF. Cotransfection with p21WAF1 restored the ability of NAB2-overexpressing PC12 cells to differentiate in response to NGF.

Abstract Decreased numbers of mast cells and abnormalities in the phenotype of mast cells are observed in the skin of mi/mi mutant mice. Recently, the mi locus was identified to encode a novel member of the basic-helix- loop-helix-leucine zipper protein family of transcription factors. Since nerve growth factor (NGF) has been reported to influence the proliferation and the phenotype of cultured mast cells (CMCs), we compared the effect of NGF between mi/mi and control normal (+/+) CMCs. Addition of NGF to the suboptimal dose of recombinant murine interleukin-3 (rmIL-3) increased the plating efficiency of +/+ CMCs, but not of mi/mi CMCs. Although +/+ CMCs were berberine sulfate- negative when cultured with rmIL-3 alone, +/+ CMCs became berberine sulfate-positive when cultured in the presence of both rmIL-3 and NGF, which suggests increased heparin content. In contrast, NGF did not influence the phenotype of mi/mi CMCs. +/+ CMCs significantly bound 125I-NGF, but mi/mi CMCs did not, which suggests a defect of NGF receptors in mi/mi CMCs. Both p75 and p140 molecules are known to be involved in the formation of NGF receptors. Although the expression of p140 messenger (m)RNA was comparable between +/+ and mi/mi CMCs, the expression of p75 mRNA was significantly lower in mi/mi CMCs than in +/+ CMCs. Taken together, the poor response of mi/mi CMCs to NGF appeared to be attributable to the impaired transcription of the p75 gene.

Decreased numbers of mast cells and abnormalities in the phenotype of mast cells are observed in the skin of mi/mi mutant mice. Recently, the mi locus was identified to encode a novel member of the basic-helix- loop-helix-leucine zipper protein family of transcription factors. Since nerve growth factor (NGF) has been reported to influence the proliferation and the phenotype of cultured mast cells (CMCs), we compared the effect of NGF between mi/mi and control normal (+/+) CMCs. Addition of NGF to the suboptimal dose of recombinant murine interleukin-3 (rmIL-3) increased the plating efficiency of +/+ CMCs, but not of mi/mi CMCs. Although +/+ CMCs were berberine sulfate- negative when cultured with rmIL-3 alone, +/+ CMCs became berberine sulfate-positive when cultured in the presence of both rmIL-3 and NGF, which suggests increased heparin content. In contrast, NGF did not influence the phenotype of mi/mi CMCs. +/+ CMCs significantly bound 125I-NGF, but mi/mi CMCs did not, which suggests a defect of NGF receptors in mi/mi CMCs. Both p75 and p140 molecules are known to be involved in the formation of NGF receptors. Although the expression of p140 messenger (m)RNA was comparable between +/+ and mi/mi CMCs, the expression of p75 mRNA was significantly lower in mi/mi CMCs than in +/+ CMCs. Taken together, the poor response of mi/mi CMCs to NGF appeared to be attributable to the impaired transcription of the p75 gene.

Nerve growth factor (NGF) treatment of PC12 cells leads to the elaboration of a neuronal phenotype, including the induction of neuronally expressed genes such as vgf. To study vgf transcription, we have created chimeric vgf/beta-globin genes in which vgf promoter sequences drive the expression of the beta-globin reporter gene or of a chimeric beta-globin gene fused to 3' untranslated vgf gene sequences. We have found that the level of inducibility of the latter construct by NGF resembles that of the endogenous vgf gene. Using transient transfection of the chimeric reporter genes into PC12 cells, into PC12 subclones expressing activated or dominantly interfering mutant Ras proteins, and into PC12 variants expressing specific NGF receptor/Trk mutants, we show that transcriptional regulation of the vgf promoter by NGF is mediated through a Ras-dependent signaling pathway. By mutational analysis of the vgf promoter, we have identified three promoter elements involved in mediating transcriptional induction by NGF and Ras. In addition to the cyclic AMP-responsive element (CRE), which binds to ATF-1, ATF-2, and CRE-binding protein in PC12 nuclear extracts, a novel CCAAT element and its binding proteins were identified, which, like the CRE, is necessary but not sufficient for the Ras-dependent induction of the vgf gene by NGF. We also identify a G(S)G element unusually located between the TATA box and transcriptional start site, which binds the NGF- and Ras-induced transcription factor, NGFI-A, and amplifies the transcriptional response. Integrating data from studies of vgf promoter regulation and NGF signal transduction, we present a model for vgf gene induction in which transcriptional activation is achieved through the persistent, direct activation of multiple interacting transcription factors binding to CRE and CCAAT elements, coordinated with the delayed transcription factor action at a G(S)G element resulting from the induced expression of NGFI-A.

To define better the function of Nerve Growth Factor (NGF) in breast cancer progression, we investigated whether this polypeptide was able to induce breast cancer cell invasion. NGF inhibited aggregation of tumour cells through modulation of the E-cadherin/catenin complex function. In addition, NGF induced the breast cancer cells to invade into Matrigel. We focused our attention on how NGF prevents aggregation, in order to discover the signalling pathway that leads tumour cells to acquire the invasive phenotype. Moreover, studies on the identification of signalling pathways that are responsive for NGF-induced invasion will be basically described.

Phaeochromocytoma PC12 cells treated with nerve growth factor (NGF) differentiate into a neuronal phenotype. Here we compare the uptake of transferrin-bound and non-transferrin-bound iron in NGF-treated (neuronal phenotype) and control (proliferating) PC12 cells. The non-transferrin-bound iron uptake was greater in the NGF-treated cells than in the control, independently of the uptake time, the iron-chelating agents used, the oxidation state of iron (Fe2+ or Fe3+) and the iron concentration tested. The NGF-treated cells expressed L-type and N-type voltage-operated Ca2+ channels. Nitrendipine (an L-type inhibitor) and possibly ω-conotoxin (an N-type inhibitor) inhibited the iron uptake by 20%. Thapsigargin inhibits the endoplasmic reticulum Ca2+ pump and allowed Mn2+ entry into cells. Preincubating PC12 cells with thapsigargin increased the iron uptake. The rate of transferrin-bound iron uptake was less than 1% of the non-transferrin-bound iron uptake and the maximum transferrin-bound iron uptake was also very low. We conclude that an increase in the iron uptake by multiple pathways accompanies the transition of PC12 cells from the proliferating to the neuronal phenotype.

Differential screening of cDNA libraries was used to detect and prepare probes for mRNAs that are regulated in PC12 rat pheochromocytoma cells by long-term (2-week) treatment with nerve growth factor (NGF). In response to NGF, PC12 cells change from a chromaffin cell-like to a sympathetic-neuron-like phenotype. Thus, one aim of this study was to identify NGF-regulated mRNAs that may be associated with the attainment of neuronal properties. Eight NGF-regulated mRNAs are described. Five of these increase 3- to 10-fold and three decrease 2- to 10-fold after long-term NGF exposure. Each mRNA was characterized with respect to the time course of the NGF response, regulation by agents other than NGF, and rat tissue distribution. Partial sequences of the cDNAs were used to search for homologies to known sequences. Homology analysis revealed that one mRNA (increased 10-fold) encodes the peptide thymosin beta 4 and a second mRNA (decreased 2-fold) encodes tyrosine hydroxylase. Another of the increased mRNAs was very abundant in sympathetic ganglia, barely detectable in brain and adrenals, and undetectable in all other tissues surveyed. One of the decreased mRNAs, by contrast, was very abundant in the adrenals and nearly absent in the sympathetic ganglia. With the exception of fibroblast growth factor, which is the only other agent known to mimic the differentiating effects of NGF on PC12 cells, none of the treatments tested (epidermal growth factor, insulin, dibutyryl cyclic AMP, dexamethasone, phorbol ester, and depolarization) reproduced the regulation observed with NGF. These and additional findings suggest that the NGF-regulated mRNAs may play roles in the establishment of the neuronal phenotype and that the probes described here will be useful to study the mechanism of action of NGF and the development and differentiation of neurons.

Microglia are the resident immune cells of the Central Nervous System (CNS). Beside classic in ammatory activities shared with macrophages, microglia actively participate in activity-dependent plasticity and learning processes [1] [2], as sculpting the neuronal circuitry during development [3] [4]. Microglia have been shown to be key players in the pathogenesis and progression of many neurodegenerative disorders and they are responsible for brain homeostasis and monitor the brain environment with their ever-moving processes [5] [6]. However, their role, either promoting or preventing pathology, is debated. On one hand, excessive activation of microglia leads to oxidative stress, neuroinammation, and eventually neuronal death [7]. On the other hand, microglial activation might be harnessed to carry out protective activities in the brain, such as phagocytosis of aggregates, synaptic pruning and formation, and the maintenance of healthy neuronal circuits [8]. Therefore, it is important to identify and modulate selectively the neuroprotective activities of microglia. The idea of microglia cells as the natural scavengers of the brain becomes especially interesting when dealing with diseases with the loss of proteostasis such as Alzheimer's disease. In the search of neuroprotective agents against neurodegeneration, neurotrophins have been historically considered as potential therapeutic candidates but usually with actions targeted to natural neuronal population. In this thesis I tested the hypothesis that microglia represent a new target cell for Nerve Growth Factor (NGF) in the brain. So far sparse experiments in the literature suggest this insight. In the literature microglia cells are known to be a source of neurotrophins [9] [10][11], most notably the Brain Derived Neurotrophic Factor (BDNF) which has been shown to promote synapse formation [1] and NGF [12] [13]. However, the extent of the modulation NGF might exert on physiological microglial functions and how this effect might come into play in neurodegenerative disorders has not been in- vestigated yet. Indeed, the main cellular targets of the neurotrophin Nerve Growth Factor (NGF) [14] in the central nervous system are considered to be the cholinergic neurons of the basal forebrain (BFCNs) [15], while its sources are mainly cortical and hippocampal neurons [16]. Consistently, interference with NGF signaling (trkA-NGF signalling) in the adult brain leads to de cits of the cholinergic system that has been related to the mechanisms driving neurodegeneration, as in the AD11 transgenic mouse model [17] [18]. The expression of anti-NGF antibodies selectively neutralizing mature NGF in the adult brain determines a progressive comprehensive neurodegeneration with neuroinflammation as the earliest observed change, at a presymptomatic phase [19] [20]. A similar progressive neurodegeneration is observed in trans- genic mice expressing a neutralizing anti TrkA antibody in the adult brain [21]. Changes in NGF homeostasis in the brain, with particular regard to the ratio of NGF to proNGF levels, have also been linked to Alzheimer's disease [22]. However the overall neurodegenerative picture induced by anti- NGF or anti-TrkA antibodies in those transgenic models is much broader than what one would expect on the basis of an action of the antibodies on the BFCNs exclusively. Moreover, the loss of NGF-TrkA signaling in the CNS, obtained by conditionally deleting NGF or TrkA genes in CNS cells derived from nestin-positive cells, has proven not to be sufficient in inducing severe cognitive impairments or neurodegen- eration in mice [23]. Altogether, this body of results has motivated our search for non neuronal targets of NGF in the adult brain. Microglia was a strong candidate, because (1) previous work had suggested that NGF could modulate some aspects of microglial cells in culture [12] and (2) transcriptomic studies in the AD11 mouse model expressing anti-NGF had shown that neuroinammation is the earliest phenotypic alteration, already at a presymptomatic phase (1 month of age; [19] [20]). In this thesis I show that microglia cells are true target of NGF both in vivo and in vitro and that the activity carried out by this neurotrophin on these myeloid cells might result neuroprotective in the context of Alzheimer's Disease.

The trigeminal ganglion (TG) is a complex sensory structure and multiple lines of evidence suggest that significant differences exist in anatomical, neurochemical and physiological properties between it and its equivalent structure in the somatosensory system, the dorsal root ganglion (DRG). This is likely to be a reflection, first on the unique areas of tissue innervation of the TG and second, on the unusual responses to injury which give rise to distinct pain symptoms such as toothache, migraine and temporomandibular joint disorders. In an attempt to address this disparity in knowledge, we have carried out an in-depth in vivo study investigating neurochemical populations and cell size distributions of sensory neurons within the rat TG. We have performed a detailed analysis of expression patterns for receptor components of important inflammatory mediators, NGF (TrkA), TNFα (p55) and IL-6 (gp130), along with the thermo-transducers TRPV1 and TRPM8. For each analysis we have compared our findings with those of the rat DRG. We have shown a significantly larger population of NF200+ neurons within the TG (51%) compared to the DRG (40%), and most interestingly, the majority of NF200+ neurons in the TG were within the small to medium cell size range, conferring a nociceptive phenotype. We have for the first time, determined expression of p55 and gp130 protein levels within neurochemically defined subpopulations of the TG. We show that a large proportion (33%) of TG neurons, in particular 27% of NF200+ neurons co-express p55, and thereby have the potential to respond directly to TNFα. Furthermore, we have observed gp130 protein expression to be ubiquitous within the TG, suggesting all neurons, including non-nociceptors, could respond to IL-6. In addition, we have utilised biochemical and electrophysiological techniques in vitro to measure the functional outcome of exposure of TG neurons to IL-6. We have demonstrated that IL-6 activates the JAK/STAT signalling pathway, preferentially within NF200+ neurons. Furthermore, we have shown that IL-6 sensitises the response of TG neurons to the TRPV1 agonist capsaicin, altering the gating properties and prolonging the opening time of the channel. Taken together, our findings support the emerging picture of a complex combinatorial pattern of co-expression of sensory neurochemicals, transducers and receptor components that help to define TG neuronal modality and function. We would advocate caution in making generalisations across sensory ganglia in particular in extrapolating data from the DRG to the trigeminal ganglion.

Abstract Background: According to the World Health Organization, 39 million people is completely blind. Between the genetic disorders causing visual impairment, by affecting 1 person to 3000-4000, Inherited Retinal Dystrophies, Occult Macular Dystrophy and Age-related Macular Degeneration are the most diffuse. Inherited Retinal Disorders (IRD) are retinal degenerations caused by mutations in at least 280 genes and more loci. IRD can be both syndromic or not-syndromic, inherited or sporadic, and all the inheritance patterns are possible. In fact, in addition to the autosomal dominant, autosomal recessive and the X-linked forms, sporadic, digenic and mitochondrial ones are possible. One of the most significant characteristics is their great genetic heterogeneity, due to the high number of genes and possible mutations involved. These genes transduce proteins involved in phototransduction and visual cycle processes, expressed in retinal pigmented epithelium or photoreceptors cells. The diseases are progressive and characterized by difficulties in night vision, loss of peripheral vision and photophobia, until complete blindness. Besides the genetic heterogeneity, these pathologies are characterized by allelic and clinical heterogeneity, which make the clinical diagnosis and the genotype-phenotype correlations complex, whenever the genetic cause is known. If genetic heterogeneity is determined by the presence of different mutations that induce the same clinical manifestation, clinical (or allelic) heterogeneity occurs in the presence of mutations on the same gene that cause different phenotypes. While Occult Macular Dystrophy (OMD) is an inherited macular dystrophy caused by mutation in RP1L1 gene with an autosomal dominant pattern of inheritance, Age-related Macular Degeneration (AMD) is a complex disorder caused by an association of genetic, environmental and advanced age susceptibility. Currently no diagnostic tests or definitive treatment for these disorders are available. Aim of the study: The objective of this project is the development of diagnostic tests valid for the genetic characterization of patients affected by IRD or OMD. For this purpose, 620 patients and affected family members were clinically characterized, and a genetic biobank was created from the collection of their DNA, available for possible genetic testing. Based on the pathology they are affected, diagnostic tests were performed using Sanger sequencing or Next Generation Sequencing (NGS), following which correlations between the genotype and the phenotype were made. Materials and methods: IRD patients were recruited and DNA extracted from blood samples. Retinal tissue and biopsies were collected too, in accordance with the current legal regulations. Genetic screening of 190 patients was realized using NGS targeted technology by MiSeq Illumina and identified mutations were then confirmed using Sanger sequencing. Genotype-phenotype correlations were elaborated, and risk factors determination was performed in 118 patients by means TaqMan PCR for 7 polymorphisms. OMD patients were screened for RP1L1 gene and 60 proband suspected to be affected by X-linked retinitis pigmentosa for RP2 and RPGR genes using Sanger sequencing. After a clinical anamnesis and in-depth visual examination, the patients were subjected to blood sampling from which the DNA was extracted and stored in the biobank. In parallel, a retinal biobank was generated from the collection of donors' retinas, collected according to the regulations in force. Genetic screening was performed on 190 patients affected by different autosomal recessive or dominant forms of IRD, using the targeted NGS technology, with panels of 25 genes. After selecting the variants or mutations identified by the analysis, Sanger sequencing was performed to confirm them. The correct attribution of the results to the analyzed individuals was achieved by comparing the genetic profiles obtained from the biobank DNA and the residual one from the NGS analysis. Genetic screening of patients with X-linked forms of IRD (n = 60) or OMD (n = 5) was obtained by means of Sanger sequencing of RPGR and RP2 (IRD) or RP1L1 (OMD) genes. A genetic susceptibility tool was developed and applied to 118 patients (39 of whom previously screened with NGS), using real time PCR, to evaluate the expression of 7 polymorphisms associated with AMD in subjects affected by IRD. Results: With NGS screening 256 variants/mutations were identified in 126 patients. Among these, 49% is represented by novel alterations. The most mutated genes are USH2A (15%) and ABCA4 (14%), while no alteration was found in the BEST1, CRX, LRAT and RLBP1 genes. 27% of cases were completely resolved, while 39% only partially, due to the identification of only one recessive mutation or of variants of uncertain significance. Missense variants cover 80% of the total, followed by nonsense mutations (9%), frameshift insertions/deletions (8%), in-frame insertions/deletions (2%) and splicing alterations (1%). The correlation of the identified genotype with the diagnosed phenotype was possible in about half of genetically characterized patients. From the comparison it was possible to identify triallelic forms, often with the missense G1961E mutation of ABCA4 as an aggravating allele, and a new gene-disease association for the PRPF31 gene that in three patients causes Usher's syndrome. The analysis of the probands with X-linked forms of Retinitis Pigmentosa allowed the identification of a frameshift insertion in the ORF15 of the RPGR gene, a nonsense mutation in RPGR gene and an already known missense mutation in RP2, in three distinct families. Among the patients with OMD, the missense S1199F mutation in RP1L1 was identified for two of them. The susceptibility test for the 7 SNPs showed a high genetic high of AMD in 38 subjects, medium high in 16, medium in 36, and low in 28. Conclusions: NGS technology has proved to be a useful diagnostic tool for the autosomal dominant or autosomal recessive forms of retinal dystrophies, allowing massive and parallel sequencing of many genes and patients. The percentage of genetically characterized patients is consistent with the data in the literature. The use of a panel of genes turns out to be an acceptable compromise between the results obtained and the criticality related to the large amount of output data, compared to whole genome or whole exome sequencing. Much of the genetic data has been correlated with the clinical manifestations of the patients. Sanger technology, gold standard of sequencing, is optimal for the identification of the genetic cause in all pathologies where one or a few genes are involved, such as the X-linked forms of IRD or the OMD. The expression of risk factors, predisposing to macular degeneration, while not correlating with the severity of the confirmed mutations, could provide important relationships with the pathologies of which the subjects are affected. The identification of the genetic causes of these pathologies will bring a new thrust to the development of gene and cell therapies, fundamental for the treatment of hereditary retinal diseases.
Riassunto Presupposti dello studio: Secondo l’organizzazione mondiale della sanità, 39 milioni di persone sono ciechi. Tra principali cause genetiche che inducono compromissione visiva, con una prevalenza di 1:2500-4000, le Distrofie Retiniche Ereditarie (i.e. Inherited Retinal Dystrophy - IRD), la Distrofia Maculare Occulta (OMD) e la Degenerazione Maculare legate all’Età (AMD) sono le più diffuse. Le prime sono un gruppo di patologie che inducono degenerazioni retiniche indotte da mutazione in almeno 280 geni e molti loci. Le forme di IRD possono essere sindromiche o non sindromiche, sporadiche o ereditarie, con tutti i pattern di eredità possibili. Infatti, oltre alle forme autosomiche dominati e recessive e le forme legate al cromosoma X, sono possibili forme sporadiche, digeniche e mitocondriali. Una delle principali caratteristiche di queste patologie è la loro elevata eterogeneità, legata all’elevato numero di geni e di mutazioni coinvolti. Questi geni, infatti, codificano proteine coinvolte nei processi di foto-trasduzione e del ciclo visivo, espressi principalmente nelle cellule dell’epitelio pigmentato retinico o nei fotorecettori. Queste malattie hanno un andamento progressivo che inizia, generalmente, con difficoltà nella visione notturna, perdita della visione periferica e fotofobia, fino alla cecità assoluta. Oltre alla eterogeneità genetica, queste patologie sono caratterizzate da eterogeneità allelica e clinica, che rendono complessa la diagnosi clinica ed anche le correlazioni genotipo-fenotipo, qualora si conosca la causa genetica del paziente. Se l‘eterogeneità genetica è determinata dalla presenza di differenti mutazioni che inducono la stessa manifestazione clinica, l’eterogeneità clinica (o allelica) si ha in presenza di mutazioni sullo stesso gene che causano fenotipi differenti. Mentre la Degenerazione Maculare Occulta è una distrofia maculare ereditaria causata da mutazioni nel gene RP1L1, con una trasmissione autosomica dominate, la Degenerazione Maculare legata all’Età è una patologia complessa causata dall’associazione di fattori genetici, ambientali e dall’età avanzata. Attualmente per queste patologie non sono disponibili né test diagnostici né cure. Scopo dello studio: L’obiettivo di questo progetto è lo sviluppo di test diagnostici validi per la caratterizzazione genetica dei pazienti affetti da IRD o OMD. Per tale scopo, 620 pazienti e familiari affetti sono stati clinicamente caratterizzati e dalla collezione dei loro DNA è stata creata una biobanca genetica disponibile per eventuali test genetici. In base alla patologia di cui sono affetti, i test diagnostici sono stati eseguiti mediante utilizzo di Sanger sequencing o Next Generation Sequencing (NGS) al seguito del quale sono stati realizzate delle correlazioni tra il genotipo e il fenotipo. Materiali e metodi: Dopo anamnesi clinica e approfondito esame visivo, i pazienti sono stati sottoposti a prelievo di sangue da cui è stato estratto il DNA stoccato all’interno della biobanca. In parallelo una biobanca retinica è stata generata dalla collezione di retine di domatori, raccolte in accordo alle normative vigenti. Lo screening genetico è stato eseguito su 190 pazienti affetti da differenti forme autosomiche recessive o dominanti di IRD, mediante la tecnologia targeted NGS, con pannelli di 25 geni. Dopo la selezione delle varianti o mutazioni identificate dall’analisi, è stato eseguito il sequenziamento Sanger per la loro conferma. La corretta attribuzione dei risultati agli individui analizzati è stata realizzata grazie alla comparazione di profili genetici ottenuti dal DNA della biobanca e quello residuo dall’analisi NGS. Lo screening genetico, dei pazienti affetti da forme X-linked di IRD (n=60) o da OMD (n=5) è stato ottenuto da sequenziamento Sanger dei geni RPGR e RP2 (IRD) o RP1L1 (OMD). Un test di suscettibilità genetica è stato sviluppato e applicato a 118 pazienti (39 dei quali precedentemente screenati con NGS), mediante real time PCR, per valutare l’espressione di 7 polimorfismi associati all’AMD nei soggetti affetti da IRD. Risultati: Con lo screening NGS 256 varianti/mutazioni sono state identificate in 126 pazienti. Tra queste il 49% è rappresentato da alterazioni non note in letteratura. I geni maggiormente mutati sono USH2A (15%) e ABCA4 (14%), mentre nessuna alterazione è stata riscontrata nei geni BEST1, CRX, LRAT and RLBP1. Il 27% dei casi è stato completamente risolto, mentre il 39% solo parzialmente, a causa dell’identificazioni di una sola mutazione recessiva o di varianti di significato incerto. Le varianti missenso coprono l'80% del totale, seguite dalle mutazioni nonsenso (9%), dalle inserzioni/delezioni frameshift (8%), dalle inserzioni/delezioni in-frame (2%) e dalle alterazioni di splicing (1%). La correlazione del genotipo identificato con il fenotipo diagnosticato è stata possibile in quasi la metà dei pazienti geneticamente caratterizzati. Dalla comparazione è stato possibile identificare forme trialleliche, con la mutazione missenso G1961E di ABCA4 come allele aggravante, e una nuova associazione gene-malattia per il gene PRPF31 che in tre pazienti causa la sindrome di Usher. L’analisi dei probandi affetti da forme X-linked di Retinite Pigmentosa ha permesso l’identificazione di un’inserzione framshift nell’ORF15 del gene RPGR, una mutazione nonsenso in RPGR gene e una mutazione missenso nota in RP2, in tre famiglie distinte. Tra i pazienti affetti da OMD, è stata identificato per due di essi la mutazione missenso S1199F in RP1L1. Il test di suscettibilità per i 7 SNP ha evidenziato un elevato alto genetico di AMD in 38 soggetti, medio alto in 16, medio in 36, e basso in 28. Conclusioni: La tecnologia NGS si è rivelata un utile strumento diagnostico per le forme autosomiche dominanti o recessive di distrofie retiniche, permettendo il sequenziamento massivo e parallelo di molti geni e pazienti. La percentuale di pazienti geneticamente caratterizzati è coerente con i dati presenti in letteratura. L’uso di un pannello di geni risulta essere un accettabile compromesso tra la i risultati ottenuti e le criticità legate alla grande quantità di dati output, rispetto alle analisi di whole genome o whole exome sequencing. Buona parte dei dati genetici sono stati correlati alle manifestazioni cliniche dei pazienti. La tecnologia Sanger, gold standard del sequenziamento, è ottimale per l’identificazione della causa genetica in tutte le patologie ove uno o pochi geni sono coinvolti, come le forme X-linked di IRD o la OMD. L’espressione di fattori di rischio, predisponenti alla degenerazione maculare, pur non correlando con la severità delle mutazioni confermate, potrebbe fornire importanti relazioni con le patologie di cui i soggetti sono affetti. L'identificazione delle cause genetiche di queste patologie porterà una nuova spinta nello sviluppo di nuove terapie geniche e cellulari, fondamentali per il trattamento delle patologie retiniche ereditarie.

Il lavoro che ho svolto durante il mio corso di dottorato è stato finalizzato a identificare e caratterizzare i geni-malattia associati a rari disturbi neurologici. In particolare ho lavorato sulla caratterizzazione fenotipica e molecolare di due pazienti affetti da una distrofia neuroassonale atipica, in cui una mutazione omozigote all'interno del gene TFG è stata identificata mediante la tecnologia WES (whole exome sequencing). Inoltre, ho anche studiato tre famiglie non correlate con individui affetti da sindrome di Leigh portatori stesse mutazioni bialleliche nel gene NDUFAF6. L'identificazione di una nuova variante intronica è stata integrata con la sua validazione funzionale attraverso l'analisi dell'mRNA. Ho anche descritto due casi con fenotipi clinici neurodegenerativi complessi. La caratterizzazione fenotipica è stata integrata con l’identificazione di due nuove mutazioni in geni-malattia già riportati, rispettivamente DNMT1 e OTX2. Nel paziente con mutazione OTX2, la presentazione molecolare e clinica non è interamente spiegata da una singola mutazione genica e sono stati identificati ulteriori possibili modificatori genetici. Questo caso rappresenta un esempio di come la raccolta dettagliata dei dati clinici e della anamnesi familiare in parallelo all'analisi dei dati NGS sia spesso utile per identificare genotipi compositi a volte associati a sindromi ereditarie complesse. Inoltre, in qualità di partner della rete internazionale europea per le malattie mitocondriali, sono stata anche coinvolto nel progetto GENOMIT; in questo contesto, ho dedicato gli ultimi mesi del mio dottorato di ricerca a studiare la praticabilità di una promettente terapia genetica xenotopica per la sindrome di Leigh e altre condizioni neurologiche associate al deficit del complesso I mitocondriale, utilizzando fibroblasti di pazienti geneticamente modificati come modello sperimentale di malattia.
The work I carried out during my PhD studies has been aimed to identify and characterize disease genes associated with rare neurological disorders. In particular I worked on phenotypic and molecular characterization of two patients with an atypical neuroaxonal dystrophy presentation, in which a homozygous mutation within the TFG gene has been identified by mean of WES (whole exome sequencing) technology. Besides, I also studied three unrelated families with individuals affected by Leigh syndrome and carrying the same biallelic mutations in the NDUFAF6 gene. The identification of a novel intronic variant has been integrated with its functional validation through mRNA analysis. I also described two cases with complex clinical neurodegenerative phenotypes. Phenotypic characterization has been integrated the identification of two novel mutations in already reported disease genes, respectively DNMT1 and OTX2. In the patient with OTX2 mutation, molecular and clinical presentation remains not entirely explained by a single gene mutation and additional possible genetic modifiers were found. This case represents an example of how detailed collection of clinical data and family history in parallel to NGS data analysis is often helpful in order to identify composite genotypes sometimes associated with complicated inherited syndromes. Additionally, as a partner of the European international network for mitochondrial disorders, I was also involved in the GENOMIT project; within this framework, I dedicated the last few months of my PhD to investigate the feasibility of a promising xenotopic genetic therapy for Leigh syndrome and other neurological conditions associated with mitochondrial complex I deficiency, using engineered patient fibroblasts as a cellular model of disease.

Collagen VI is a large extracellular matrix protein with a widespread distribution in several tissues. Mutations of genes encoding for collagen VI in humans cause various forms of muscular dystrophies, including Bethlem Myopathy (BM) and Ullrich Congenital Muscular Dystrophy (UCMD). Previous studies demonstrated that collagen VI null (Col6a1–/–) mice display an early onset myopathic phenotype affecting skeletal muscles, characterized by mitochondrial dysfunction, and impaired autophagy. Molecular pathway(s) transducing collagen VI signals are largely unknown. NG2 is a transmembrane proteoglycan acting as a cell surface receptor for collagen VI. NG2 is not only expressed by developing tissues and tumors but also by postnatal skeletal muscles and it is selectively down-regulated in myofibers from UCMD patients and Col6a1–/– mice. The first aim of my PhD work was to investigate the role of NG2 and its interplay with collagen VI in skeletal muscles. Although NG2 null (Cspg4–/–) mice appear phenotypically normal, careful investigation of muscles revealed that these mice are affected by a myopathic syndrome. In particular, Cspg4–/– myofibers showed increased incidence of apoptosis and dysfunctional mitochondria. When Cspg4–/– animals were subjected to voluntary exercise on running wheel, they displayed muscle weakness and decreased resistance to fatigue. Electron microscopy of Cspg4–/– mice revealed increased thickness of basement membrane and proliferation of pericytes in the capillary vessels of skeletal muscles. These ultrastructural alterations are remarkably similar to those detected in Congenital Myosclerosis, a rare human disorder recently linked to a peculiar COL6A2 mutation. These findings suggest that NG2 may influence pericyte homeostasis contributing to muscle fiber degeneration in collagen VI related diseases. Thus, the phenotype of NG2 null mice is partially similar to that of collagen VI null mice, pointing at an interaction between these two molecules in skeletal muscles. The second aim of my PhD work was to evaluate the therapeutic efficacy of NIM811, a non-immunosuppressive cyclosporin A derivative, in Col6a1–/– mice and in muscle cultures from patients affected by UCMD and BM. The results indicate that in vivo treatment of Col6a1–/– mice with NIM811 is very effective at normalizing the mitochondrial phenotype in muscle fibers and at reverting the apoptotic rate to a frequency close that observed in wild-type animals. Interestingly, 10 days of treatment with NIM811 showed a marked recovery of muscle strength in Col6a1–/– mice. Moreover, treatment with NIM811 delayed mitochondrial depolarization induced by both oligomycin and rotenone in muscle cultures from UCMD and BM patients. Considering the results obtained with NIM811 in Col6a1–/– mice and in patient cultures, that this drug has no immunosuppressive side-effect and that it is available, NIM811 appears very promising for clinical trial of both pediatric and adult patients affected by collagen VI myopathies.
Il collagene VI è una proteina della matrice extracellulare ampiamente distribuita in diversi tessuti. Le mutazioni a carico dei geni che lo codificano sono causa di diverse forme di distrofia muscolare umana, ed in particolare della miopatia di Bethlem (BM) e della distrofia muscolare congenita di Ullrich (UCMD). Studi precedenti hanno dimostrato che i topi privi di collagene VI (Col6a1–/–) mostrano un fenotipo miopatico ad esordio precoce, che interessa i muscoli scheletrici e che è caratterizzato da disfunzione mitocondriale ed alterazione dell’autofagia. Le vie molecolari che trasducono i segnali del collagene VI sono per lo più sconosciute. NG2 è un proteoglicano transmembrana che funge da recettore di superficie per il collagene VI. Esso è espresso non solo nei tessuti in via di sviluppo e nei tumori ma anche nei muscoli scheletrici adulti ed è selettivamente down-regolato nelle miofibre di pazienti affetti da UCMD e nei topi privi di collagene VI. Il primo obiettivo del mio dottorato è stato investigare il ruolo di NG2 e la sua relazione con il collagene VI nei muscoli scheletrici. Sebbene il topo privo di NG2 (Cspg4–/–) appaia fenotipicamente normale, un’attenta indagine sui muscoli ha rivelato che questi animali sono affetti da una sindrome miopatica. In particolare, le miofibre dei topi privi di NG2 hanno mostrato un’aumentata incidenza di eventi apoptotici e di mitocondri disfunzionanti. Quando i topi Cspg4–/– sono stati sottoposti ad esercizio volontario su ruote, hanno manifestato debolezza muscolare e ridotta resistenza alla fatica. La microscopia elettronica ha evidenziato un ispessimento della lamina basale dei vasi ed una proliferazione di periciti attorno a capillari dei muscoli scheletrici. Queste alterazioni ultrastrutturali sono assai simili a quelle riscontrate nei pazienti affetti da miosclerosi congenita, una rara malattia recentemente associata ad una particolare mutazione nel gene COL6A2. Questa scoperta suggerisce che NG2 potrebbe influenzare l’omeostasi dei periciti contribuendo alla degenerazione delle fibre muscolari in disordini associati ad alterazioni del collagene VI. Il fenotipo osservato nei topi privi di NG2 è quindi parzialmente simile a quello osservato nei topi privi di collagene VI, rafforzando l’ipotesi di un’interazione tra le due molecole nei muscoli scheletrici. Il secondo obiettivo del mio elaborato di tesi è stato valutare l’efficacia terapeutica di NIM811, un derivato non immunosoppressivo della ciclosporina A, in topi privi di collagene VI e in colture derivate da biopsie muscolari di pazienti affetti da UCMD e BM. Il trattamento in vivo con NIM811 nei topi Col6a1–/– è risultato essere molto efficace nel normalizzare il fenotipo mitocondriale nelle miofibre e nel riportare l’apoptosi a livelli confrontabili con i topi selvatici. È anche interessante osservare che 10 giorni di trattamento con NIM811 hanno permesso un significativo recupero della forza muscolare nei topi Col6a1–/–. Inoltre, il trattamento con NIM811 ha ridotto la depolarizzazione mitocondriale indotta da oligomicina e rotenone nelle culture muscolari da pazienti affetti da UCMD e BM. Considerando i risultati ottenuti con NIM811 in topi privi di collagene VI e culture da pazienti, l’effetto non immunosoppressivo e la disponibilità del farmaco, NIM811 appare davvero promettente per un trial clinico sia su pazienti pediatrici che adulti affetti da disordini muscolari associati ad alterazioni del collagene VI.

L’anémone de mer symbiotique Anemonia viridis possède cinq morphes définis à l’aide de critères morphologiques. Premièrement, le statut taxonomique de trois des morphes d’A. viridis (var. rufescens, rustica et smaragdina) a été précisé à l’aide de marqueurs moléculaires basés sur des gènes de stress et des marqueurs RAD. Nous avons pu déterminer que ces trois morphes ne formaient qu’une seule espèce et mis en évidence quatre lignées génétiques indépendantes sur base de la géographie (trois en mer Méditerranée, une dans la Manche). Par l’utilisation des variations des séquences ITS2, nous n’avons pu détecter aucune implication du symbiote (Symbiodinium sp.) dans la différenciation des morphes, mais nous avons révélé une composition en symbiotes divergente entre les lignées génétiques indépendantes de l’hôte animal. Par ailleurs, A. viridis se développe dans des environnements particulièrement contrastés, faisant d’elle un modèle d’étude idéal pour l’étude de l’adaptation locale chez les Cnidaires. Par conséquent, l’adaptation locale chez A. viridis a été testée en comparant des populations venant de sites aux conditions environnementales contrastées (surface vs. profondeur et lagune vs. mer). Une recherche de loci outliers sur des marqueurs RAD et des marqueurs de gènes de stress n’a toutefois révélé aucun gène candidat dans l’adaptation locale par rapport aux conditions environnementales testées. Ce travail a donc permis de définir A. viridis comme un organisme extrêmement plastique capable de posséder un fort polymorphisme intrinsèque et de s’acclimater à des habitats contrastés
The symbiotic sea anemone Anemonia viridis has five morphs described using morphological traits. First, the taxonomical status of three of the morphs of A. viridis (var. rufescens, rustica and smaragdina) was studied using stress gene markers and RAD markers. We revealed that the three morphs were not different species, but that A. viridis was split into four polymorphic independent genetic lineages based on geographical origin (three in the Mediterranean Sea, one in the English Channel). Using ITS2 sequence variation, we could not detect any implication of the symbiont (Symbiodinium sp) in the morph differentiation, but we revealed a divergence in symbiont composition among the geographic independent lineages of the animal host. If no effect of the symbiont was detected, a variable distribution of the ITS2 variants based on geography was revealed. Moreover, A. viridis lives in highly contrasted environments, making it an ideal species to study local adaptation. Thus, local adaptation was tested on A. viridis by comparing populations coming from contrasted environments (shallow vs. deep and lagoon vs. sea). Using RAD and stress genes markers in a search for outlier loci, we revealed no candidate adaptive genes under our environmental conditions. In conclusion, Anemonia viridis seems to be a very plastic organism, with a high intrinsic polymorphism and a high acclimation potential

Per lungo tempo si è pensato che i precursori delle neurotrofine servissero solo per generare la forma matura della proteina, l’unica biologicamente attiva. Recentemente è invece stato dimostrato che il proNGF è espresso in grande quantità nel cervello e che è biologicamente attivo in sistemi in vitro; tuttavia le informazioni ottenute fino ad oggi riguardo i suoi effetti sulle cellule neuronali sono poche e contrastanti. L’interesse per questa molecola, inoltre, è cresciuto da quando è stato ipotizzato un suo possibile coinvolgimento nella patogenesi della malattia di Alzheimer. In questo lavoro sono state studiate le caratteristiche e le funzioni biologiche del precursore dell’NGF comparando i suoi effetti con quelli della forma matura dell’NGF. A tal fine sono stati analizzati gli effetti delle molecole ricombinanti di proNGF WT (clivabile), MUT (resistente al taglio) e dell’NGF maturo sul fenotipo e sulla funzionalità dei neuroni colinergici del “basal forebrain”. I neuroni colinergici del “basal forebrain” sono una delle poche popolazioni neuronali ad esprimere tutti i recettori per l’NGF e per il proNGF (p75, TrkA, Sortilina) per tutta la vita. Il confronto tra gli effetti dell’NGF e delle due forme di proNGF utilizzate ha mostrato che i trattamenti non avevano effetto sulla morte e sopravvivenza neuronale e/o sulla differenziazione neuronale, avevano la stessa capacità di aumentare le dimensioni del corpo cellulare in maniera dipendente dall’attivazione del recettore TrkA, aumentavano il rilascio di Ach (misura della funzionalità colinergica) in modo dipendente dall’attivazione del p75 e di TrkA. Inoltre sia l’NGF che le due forme di proNGF aumentavano l’espressione di p75, ma attraverso l’attivazione di diversi recettori per l’NGF. Infine modulavano in maniera diversa l’espressione dei markers colinergici ChAT e CHT e l’espressione del recettore TrkA. Il proNGF MUT era l’unica delle molecole utilizzate in grado di aumentare l’espressione di TrkA. Questo risultato è particolarmente interessante in quanto mette in evidenza che la molecola potrebbe essere usata a scopo terapeutico per prevenire la diminuzione di TrkA che si osserva nelle fasi precoci della malattia di Alzheimer. In questo lavoro è stato anche dimostrato che il p75 ha un ruolo importante nella modulazione del rilascio di Ach sia costitutivo che indotto dalle neurotrofine. Ciò suggerisce che p75 potrebbe essere un target terapeutico per il controllo della riduzione della funzionalità colinergica tipica della malattia di Alzheimer. Questo studio, quindi, descrive alcune caratteristiche e funzioni biologiche del proNGF e del p75 utili per comprenderne il ruolo fisiologico ed il loro possibile coinvolgimento nell’eziopatogenesi della malattia di Alzheimer.
The pro- forms of neurotrophins have been thought for a long time as exclusively necessary for the generation of the biologically active mature proteins. Recently, it has been demonstrated that proNGF, abundantly expressed in the brain, retains a biological activity in vitro, but little and contrasting informations have been produced about its effects on neuronal cells. Growing interest has been shown for this molecule, due to its possible involvement in AD pathogenesis In this work we studied the characteristics and biological functions of the precursor form of NGF comparing its effects to that of the mature form. To this aim we analyzed the effects of recombinant proNGF WT (cleavable), MUT (resistant to cleavage) and mature NGF on phenotype and function of basal forebrain cholinergic neurons (BFCNs), one of the few neuronal populations expressing throughout life the whole complement of NGF and proNGF receptors (p75, TrkA and sortilin). The comparison between NGF and proNGFs effects showed that all treatments had no effect on neuronal death/survival and/or differentiation, but had a similar TrkA-dependent effect on the soma size increase, and a TrkA- and p75-dependent ACh release enhancement. Moreover, both NGF and proNGFs increased p75 expression, but through a different activation of p75 or TrkA. Finally, they differently affected cholinergic markers expression (ChAT and CHT) and TrkA expression. Of interest, proNGF MUT increased TrkA expression, highlighting a possible therapeutic use of the molecule in preventing the decrease of TrkA levels observed in early stages of AD. Our results also demonstrated that p75 had an important role in controlling both neurotrophin-induced and constitutive ACh release, suggesting p75 receptor as a possible therapeutic target for the prevention of cholinergic hypofunction typical of AD. In conclusion, this study describes characteristics and biological functions of proNGF and p75 useful to understand their role in physiological and their possible involvement in AD pathological conditions.

Hypertrophic cardiomyopathy (HCM) is a common genetic heart disorder characterized by unexplained left ventricle hypertrophy associated with non-dilated ventricular chambers. Several genes encoding heart sarcomeric proteins have been associated to HCM, but a small proportion of HCM patients harbor alterations in other non-sarcomeric loci. The variable expression of HCM seems influenced by genetic modifier factors and new sequencing technologies are redefining the understanding of genotype-phenotype relationships, even if the interpretations of the numerous identified variants pose several challenges.We investigated 62 sarcomeric and non-sarcomeric genes in 41 HCM cases and in 3 HCM-related disorders patients. We found that 82% of the patients harbored at least one rare nsSNV: 11% of the patients showed only sarcomere nsSNVs, 20% of cases harbored at least one sarcomeric nsSNV with at least a desmosomal one and 14% displayed at least one desmosomal nsSNV but no other sarcomere change. We reported an association between desmosomal variations and the pathogenesis of HCM that has not been described to date. We employed an integrated approach that combines multiple tools for the prediction, annotation and visualization of functional variants. Several different methods were employed to predict the functional consequences of alleles that result in amino acid substitutions, to study the effect of some variants over the splicing process and to investigate the impact of these changes respect to the evolutionary conservation. Genotype-phenotype correlations were carried out for inspecting the involvement of each gene in age onset and clinical variability of HCM. Statistical analyses revealed an inverse correlation between the number of nsSNVs and age at onset, and a relationship between the clinical variability and number and type of variants. Then, we describe the clinical, pathological, and molecular features of the novel LAMP2 c.453delT mutation in one of our HCM-related disorders patients affected by Danon disease characterized by severe hypertrophic cardiomyopathy, mild intellectual impairment and rapid progression to heart failure, requiring heart transplant. Immunohistochemical analysis of LAMP2 in the explanted heart revealed a mosaic pattern of distribution, with discrete clusters of either stained or unstained cardiac myocytes, the latter being more frequent in the septum. Interestingly, multiple foci of microscarring were found on histology in the Left Ventricle (LV) free wall and septum. Our findings suggest that several features may contribute to the early and severe cardiac phenotype in female patients affected by Danon disease. In conclusion, this work aims to extend the mutational spectrum of HCM and to contribute in defining the molecular pathogenesis and inheritance pattern(s) of this condition. Besides, we delineate a specific procedure for the identification of the most likely pathogenetic variants for a next generation sequencing approach embodied in a clinical context

The landmark paper discussed in this chapter is ‘Congenital insensitivity to pain. A clinical, genetic and neurophysiological study of four children from the same family’, published by D. C. Thrush in 1973. The study of patients with congenital conditions that result in pain insensitivity has been invaluable in helping define the molecular mechanisms of sensory processing. These patients share a major defining phenotype (they feel little or no pain from birth), although they often have differing subtle symptoms which belie a host of separate conditions that we have now started to recognize with the advent of molecular genetics (e.g. loss-of-function mutations in the gene encoding Nav1.7, and mutations related to nerve growth factor (NGF)); these include congenital insensitivity to pain with anhydrosis (CIPA; thought to be due to mutations in the gene encoding the NGF receptor NTRK1) and hereditary sensory and autonomic neuropathies (HSANs) such as familial dysautonomia.

Abstract The wide application of next-generation sequencing (NGS) has facilitated and accelerated causal gene finding and breeding in the field of plant sciences. A wide variety of techniques and computational strategies is available that needs to be appropriately tailored to the species, genetic architecture of the trait of interest, breeding system and available resources. Utilizing these NGS methods, the typical computational steps of marker discovery, genetic mapping and identification of causal mutations can be achieved in a single step in a cost- and time-efficient manner. Rather than focusing on a few high-impact genetic variants that explain phenotypes, increased computational power allows modelling of phenotypes based on genome-wide molecular markers, known as genomic selection (GS). Solely based on this genotype information, modern GS approaches can accurately predict breeding values for a given trait (the average effects of alleles over all loci that are anticipated to be transferred from the parent to the progeny) based on a large training population of genotyped and phenotyped individuals (Crossa et al., 2017). Once trained, the model offers great reductions in breeding speed and costs. We advocate for improving conventional GS methods by applying advanced techniques based on machine learning (ML) and outline how this approach can also be used for causal gene finding. Subsequent to genetic causes of agronomically important traits, epigenetic mechanisms such as DNA methylation play a crucial role in shaping phenotypes and can become interesting targets in breeding pipelines. We highlight an ML approach shown to detect functional methylation changes sensitively from NGS data. We give an overview about commonly applied strategies and provide practical considerations in choosing and performing NGS-based gene finding and NGS-assisted breeding.

AbstractMutagenesis of in vitro propagated bananas is an efficient method to introduce novel alleles and broaden genetic diversity. The FAO/IAEA Plant Breeding and Genetics Laboratory previously established efficient methods for mutation induction of in vitro shoot tips in banana using physical and chemical mutagens as well as methods for the efficient discovery of ethyl methanesulphonate (EMS) induced single nucleotide mutations in targeted genes. Officially released mutant banana varieties have been created using gamma rays, a mutagen that can produce large genomic changes such as insertions and deletions (InDels). Such dosage mutations may be particularly important for generating observable phenotypes in polyploids such as banana. Here, we describe a Next Generation Sequencing (NGS) approach in Cavendish (AAA) bananas to identify large genomic InDels. The method is based on low coverage whole genome sequencing (LC-WGS) using an Illumina short-read sequencing platform. We provide details for sonication-mediated library preparation and the installation and use of freely available computer software to identify copy number variation in Cavendish banana. Alternative DNA library construction procedures and bioinformatics tools are briefly described. Example data is provided for the mutant variety Novaria and cv Grande Naine (AAA), but the methodology can be equally applied for triploid bananas with mixed genomes (A and B) and is useful for the characterization of putative Fusarium Wilt TR4 resistant mutant lines described elsewhere in this protocol book.

AbstractIn silico prediction of plant performance is gaining increasing breeders’ attention. Several statistical, mathematical and machine learning methodologies for analysis of phenotypic, omics and environmental data typically use individual or a few data layers. Genomic selection is one of the applications, where heterogeneous data, such as those from omics technologies, are handled, accommodating several genetic models of inheritance. There are many new high throughput Next Generation Sequencing (NGS) platforms on the market producing whole-genome data at a low cost. Hence, large-scale genomic data can be produced and analyzed enabling intercrosses and fast-paced recurrent selection. The offspring properties can be predicted instead of manually evaluated in the field . Breeders have a short time window to make decisions by the time they receive data, which is one of the major challenges in commercial breeding. To implement genomic selection routinely as part of breeding programs, data management systems and analytics capacity have therefore to be in order. The traditional relational database management systems (RDBMS), which are designed to store, manage and analyze large-scale data, offer appealing characteristics, particularly when they are upgraded with capabilities for working with binary large objects. In addition, NoSQL systems were considered effective tools for managing high-dimensional genomic data. MongoDB system, a document-based NoSQL database, was effectively used to develop web-based tools for visualizing and exploring genotypic information. The Hierarchical Data Format (HDF5), a member of the high-performance distributed file systems family, demonstrated superior performance with high-dimensional and highly structured data such as genomic sequencing data.

Phelan-McDermid (PMS) or 22q13 deletion syndrome (OMIM 606232) is a rare genetic disorder with highly variable clinical presentation. The phenotype includes generalized neonatal hypotonia, developmental delay with intellectual disability and delayed speech, mild dysmorphic features, and autistic behavior. The genetic defects of PMS consist of 22q13.3 deletions or chromosomal structural rearrangements involving SHANK3 gene; the loss of function mutations of SHANK3 gene was reported in a minority of cases. The 22q13.3 deletions vary in size, from 0.2 to over 9 Mb, and, although larger deletions are generally associated with more severe phenotypes, the genotype-phenotype correlations are not clear-cut for all patients. SHANK3 is considered the main candidate gene for the neurologic features of PMS. PMS is a rare disorder, often underdiagnosed. There are no established clinical diagnostic criteria for PMS. The genetic tests typically used are chromosomal microarray and multiplex ligation-dependent probe amplification (MLPA) or fluorescent in situ hybridization (FISH) for copy number analysis of SHANK3 gene; next-generation sequencing (NGS) or Sanger sequencing is used for pathogenic mutation screening of SHANK3. In this chapter, we report three cases with PMS and summarize the clinical and genetic diagnostic approaches of this condition, highlighting the role of chromosomal microarray technology in the identification of rare, but significantly impacting patient’s life, DNA copy number abnormalities.

The chapter suggests the need of a “second-generation” candidate gene approach to adapt to first-generation limitations and to strengthen efforts to study GE interactions. The sample sizes associated with many phenotypes and areas of study in the literature (e.g., clinical trials, neuroimaging studies) are unlikely to yield sample sizes in the area of GWA and NGS studies (i.e., 200,000-300,000 participants). However, by building upon prior limitations in the candidate gene literature, using findings from GWA and NGS studies and meta-analyses, using multiple methods of analyses (e.g., gene expression analysis; methylation analysis), and using theory and prior substantive research to guide hypothesis testing, progress can be made on G X E interactions for complex phenotypes. Several illustrative path models were provided in this chapter to provide a visual frame for how we have approached G X E interactions in the past, and how, going forward, we might proceed to investigate multiple polygenic by multiple environmental models. This level of complexity may be necessary to advance the field to address the many exciting research questions of interest, as well as the challenges that confront us as we attempt to move this knowledge from discovery to practice.

Recent advances in human exome sequencing and the associated advantages have made it a technology of choice in various domains. The savings in time, cost and data storage compared with whole genome sequencing make this technology a potential game changer in clinical research settings. Recent advances in NGS have made it feasible to use exome sequencing in clinical research for identifying novel and rare variants that can lead to change in protein structure and function which may finally culminate into a totally different phenotype. If whole exome is not desired the same technology can be used for studying target exonic regions to investigate causative genes for a specific phenotype associated with disease. Exome sequencing has emerged as an effective and efficient tool for the translational and clinical research. There is a demand for systematically storing variant information in large databanks. Meaningful information from the exome-seq data can be combined with other data. This can be correlated with clinical findings within a clinical trial setting for a better study outcome.

Neurofibromatosis type 1, NF-1, is a common monogenic (NF1) disease, characterized by highly variable clinical presentation and high predisposition for tumors, especially those of astrocytic origin (low- to high-grade gliomas). Unfortunately, very few genotype–phenotype correlations have been possible, and the numerous identified mutations do not offer help for prognosis and patient counselling. Whole gene deletion in animals does not successfully model the disease, as NF-1 cases caused by point mutations could be differentially affected by cell type-specific alternative splice variants of NF1. In this chapter, we will discuss the differential Microtubule-Associated-Protein (MAP) properties of NLS or ΔNLS neurofibromins, produced by the alternatively splicing of exon 51, which also contains a Nuclear Localization Sequence (NLS), in the assembly of the mitotic spindle and in faithful genome transmission. We will also commend on the major theme that emerges about NLS-containing tumor suppressors that function as mitotic MAPs.

AbstractRegulatory RNAs like microRNAs (miRNAs) and long non-coding RNAs (lncRNAs) control vascular and immune cells’ phenotype and thus play a crucial role in atherosclerosis. Moreover, the mutual interactions between miRNAs and lncRNAs link both types of regulatory RNAs in a functional network that affects lesion formation. In this review, we deduce novel concepts of atherosclerosis from the analysis of the current data on regulatory RNAs’ role in endothelial cells (ECs) and macrophages. In contrast to arterial ECs, which adopt a stable phenotype by adaptation to high shear stress, macrophages are highly plastic and quickly change their activation status. At predilection sites of atherosclerosis, such as arterial bifurcations, ECs are exposed to disturbed laminar flow, which generates a dysadaptive stress response mediated by miRNAs. Whereas the highly abundant miR-126-5p promotes regenerative proliferation of dysadapted ECs, miR-103-3p stimulates inflammatory activation and impairs endothelial regeneration by aberrant proliferation and micronuclei formation. In macrophages, miRNAs are essential in regulating energy and lipid metabolism, which affects inflammatory activation and foam cell formation.Moreover, lipopolysaccharide-induced miR-155 and miR-146 shape inflammatory macrophage activation through their oppositional effects on NF-kB. Most lncRNAs are not conserved between species, except a small group of very long lncRNAs, such as MALAT1, which blocks numerous miRNAs by providing non-functional binding sites. In summary, regulatory RNAs’ roles are highly context-dependent, and therapeutic approaches that target specific functional interactions of miRNAs appear promising against cardiovascular diseases.

Improved fiber yield is considered a constant goal of upland cotton (Gossypium hirsutum) breeding worldwide, but the understanding of the genetic basis controlling yield-related traits remains limited. Dissecting the genetic architecture of complex traits is an ongoing challenge for geneticists. Two complementary approaches for genetic mapping, linkage mapping and association mapping have led to successful dissection of complex traits in many crop species. Both of these methods detect quantitative trait loci (QTL) by identifying marker–trait associations, and the only fundamental difference between them is that between mapping populations, which directly determine mapping resolution and power. Nowadays, the availability of genomic tools and resources is leading to a new revolution of plant breeding, as they facilitate the study of the genotype and its relationship with the phenotype, in particular for complex traits. Next Generation Sequencing (NGS) technologies are allowing the mass sequencing of genomes and transcriptomes, which is producing a vast array of genomic information with the development of high-throughput genotyping, phenotyping will be a major challenge for genetic mapping studies. We believe that high-quality phenotyping and appropriate experimental design coupled with new statistical models will accelerate progress in dissecting the genetic architecture of complex traits.

Breast cancer is the most aggressive and rapidly growing cancer worldwide. It is classified into several subtypes among which triple negative breast cancer (TNBC) is the most aggressive. Oncogene regulation leads to increased signal activity in the cells and contributes to the tumorigenic phenotype. Such signals generate a large number of proteins that provoke cell growth and inhibit cell death. Transcription factors, such as miR-27a, NF-κB, and FOXM1, are proteins that are conserved across species, abundantly found in the nucleus, bind to DNA, and enforce specific target genes. Oncogenic transcription factors arising from mutation accompanying aberrant gene expression transfer signals to the nucleus and interrupts transcription patterns and stimulation of oncogenic transcription factors consistent with cell cycle regulation, apoptosis, proliferation, and cell differentiation. This review gives a detailed framework about the treatment opportunities and highlights the importance of few transcription factors which may act as an important biomarker specifically for TNBC.

Abstract Rotating Wall Vessel (RWV) Bioreactors simulate microgravity and facilitate 3-D tissue-like assembly through spatial co-localization and cell-cell interactions. This unique cell culture venue is well suited to assess the role of micro-environmental cues in the assembly and tissue-specific differentiation of cells in culture. Our long term goal is to use RWV Bioreactors for generating functional neuroendocrine 3-D constructs which may be useful as clinical replacement tissue in treating neurodegenerative diseases. As a model we are using PC12 pheochromocytoma cells, a bipotential rat adrenal medullary tumor cell line. PC 12 cells differentiate, depending on exogenous factors, either along the neuronal or the neuroendocrine pathway. PC12 cells, when maintained for up to 20 days in RWV Bioreactors, form macroscopic tissue-like aggregates which exhibit enhanced expression of neuroendocrine, adrenergic differentiation markers (Lelkes et al., In Vitro Devel. Biol, 1998, 34: 316–325). We hypothesized that exposure of PC12 cells to the “simulated microgravity” culture conditions in the RWV Bioreactors, might selectively activate signal transduction pathways leading to enhanced neuroendocrine adrenergic differentiation. Using quantitative RT-PCR we demonstrated rapid upregulation of an adrenergic marker, phenylethanolamine-N-methyl transferase (PNMT), in short term RWV cultures. Concomitantly, we found, by electrophoretic mobility shift assays, differential induction of nuclear transcription factors, such as GRE and SP-1, which are known to be involved in the glucocorticoid-induced activation of PNMT. Conversely, upon short term culture of PC12 cells in RWV, the neuronal traits of the cells were impaired. Upon exposure to simulated microgravity, MAPK signaling (erk and jnk) was constitutively activated, while nerve-growth factor (NGF)-induced activation of erk, was abrogated. These results suggest that the culture conditions in the RWV Bioreactors are sufficient to induce PC12 cell differentiation towards the neuroendocrine, phenotype by upregulating “adrenergic” gene expression, while downregulating neurotrophin signaling pathways.

Context: Myofibrillar myopathies (MFM) represent a heterogeneous group of disorders of skeletal and cardiac muscle caused by mutations in genes that encode proteins of sarcomere. Diagnosis is a challenge due to clinical and genetic variability. Case report: Woman, 36 years old, presenting stumbles and falls for 3 years evolving with proximal limb weakness. At age 30, she fainted and a cardiac pacemaker was implanted. Non-consanguineous parents. Neurological exam: proximal and distal weakness in lower limbs and distal atrophy; osteotendinous reflexes normal. Bilateral scapula alata. Exams: CPK = 457 U / l; EMG: myopathic pattern. Muscle MRI: diffuse and heterogeneous fatty degeneration, marked in sartorius, gracilis and semitedinous. Panel NGS myopathies: pathogenic variant, c.1175T> C, missense in heterozygosis in desmin gene. CONCLUSION: The diagnosis of MFM is based on the morphological findings of muscle biopsy with the presence of protein aggregates as a determining factor. Currently, genetic testing by NGS has facilitated early diagnosis allowing for a more appropriate clinical approach. The desmin gene was the first one described to be associated with this group of myopathies. It encodes the desmin protein, a member of the intermediate filament family present in cardiac and skeletal muscle. Several phenotypes are related to desmin gene: isolated dilated cardiomyopathy; scapuloperoneal weakness and distoproximal weakness with cardiac alterations. Desminopathy is a rare cause of cardiomyopathy and / or myopathy. The diagnosis should be thought in patient with muscle weakness and cardiac changes.

Introduction: Breast cancer is the most frequent type of cancer in the world and the biggest cause of female deaths. About 10%–15% of cases are due to hereditary factors. The profile of genetic variants is still scarcely known among the Brazilian population and there are no published data for the central region of the country. Objectives: This study aimed to analyze the profile of pathogenic variants (PV) and of the ones of uncertain significance (VUS) for the RAD50, RAD51C, RAD51D, ATM, PALB2, BRIP1, BARD1 and CHEK2 genes in this population. Methods: 113 patients diagnosed with breast or ovarian cancer who met the National Comprehensive Cancer Networking criteria for hereditary breast and ovarian cancer syndrome were selected. The genes had all regions sequenced using NGS (New Generation Sequencing) and the raw data were evaluated using the Sophia DDM and IonReporter softwares. Results: A total of 3.53% of patients had PV in the PALB2 (c.2257C>T), BARD1 (c.176_177delAG), RAD50 (c.2165dupA) or ATM (c.7913G>A) genes. Patients with pathogenic variants in ATM and PALB2 genes were diagnosed before the age of 40. Patients with pathogenic variants in the BARD1 and RAD50 genes had triple negative breast cancer before the age of 60. The patient with a pathogenic variant in the RAD50 gene also developed ovarian cancer. It was observed that 24.77% of the patients had some VUS, 35.29% of which were in the ATM gene, and a new VUS in the CHEK2 gene (c.1151T>C), related to male breast cancer. Conclusions: These findings contribute to a better understanding of the phenotype of patients with pathogenic variants related to breast cancer in non-BRCA genes. In addition, it reveals a new pathogenic variant in the CHEK2 gene, not described in the literature, related to a case of male breast cancer.

The Problem: Abscission is a highly regulated process, occurring as a natural terminal stage of development, in which various organs are separated from the parent plant. In most plant species, the process is initiated by a decrease in active auxin in the abscission zone (AZ) and an increase in ethylene, and may be accelerated by postharvest or environmental stresses. Another potential key regulator in abscission is IDA (Inflorescence Deficient in Abscission), which was identified as an essential peptide signal for floral organ abscission in Arabidopsis. However, information is still lacking regarding the molecular mechanisms integrating all these regulators. In our previous BARD funded research we made substantial progress towards understanding these molecular events in tomato, and the study is still in progress. We established a powerful platform for analysis of genes for regulatory proteins expressed in AZ. We identified changes in gene expression for several transcription factors (TFs) directly linked to ethylene and auxin signaling and several additional regulatory proteins not so obviously linked to these hormones. Moreover, we demonstrated using a virus-induced gene silencing (VIGS) assay that several play a functional role in the onset of abscission. Based on these results we have selected 14 genes for further analysis in stably transformed tomato plants. All 14 genes were suppressed by RNA interference (RNAi) using a constitutive promoter, and 5 of them were also suppressed using an abscission-specific promoter. Transformations are currently at different stages of progress including some lines that already display an abscission phenotype. Objectives: We propose here to (1) complete the functional analysis of the stably transformed tomato plants with T2 lines and perform transcriptome analysis using custom abscission-specific microarrays; (2) conduct an indepth analysis of the role of IDA signaling in tomato leaf and flower abscission; (3) perform transcriptome and proteome analyses to extend the earlier gene expression studies to identify transcripts and proteins that are highly specific to the separation layer (i.e., target cells for cell separation) prior to the onset of abscission; (4) extend and compliment the work in tomato using a winnowed set of genes in soybean. Methodology: Next Generation Sequencing (NGS) of mRNA will be used to further increase the list of abscission-associated genes, and for preparation of a custom tomato abscission microarray to test altered gene expression in transgenic plants. Tandem mass spectrometry (LC-MS/MS) of protein extracts from leaf petiole, flower pedicel and their AZ tissues will be used to identify the proteome of the AZ before and during abscission. AZ-specific gene promoters will be used in stably transformed tomato plants to reduce non-target phenotypes. The bean pod mottle virus (BPMV) plasmid vectors will be used for VIGS analysis in soybean. Expected Contribution: Our study will provide new insights into the regulation of ethylene-induced abscission by further revealing the role of key regulators in the process. This will permit development of novel techniques for manipulating leaf and flower abscission, thereby improving the postharvest performance of agriculturally important crops.