Dissecting the genetic heterogeneity of familial dementias

La demenza \ue8 una sindrome clinica caratterizzata da declino cognitivo le cui principali manifestazioni comprendono la perdita di memoria, deficit del linguaggio, deterioramento delle capacit\ue0 di pianificazione e di giudizio, cambiamenti di comportamento e, non di rado, compromissione delle funzioni motorie ed infine dell'autonomia. Oggigiorno, si stima che circa 36 milioni di persone in tutto il mondo siano affette da demenza, stime che, secondo le ultime predizioni, \ue8 destinata a crescere fino a 115 milioni nel 2050. Considerando non solo la prevalenza bens\uec anche l'impatto sociale ed economico che questa malattia comporta, negli ultimi anni la ricerca scientifica sta investendo molte risorse in studi finalizzati all'identificazione delle cause e dei meccanismi patologici che sottendono a questa malattia. Lo scopo ultimo \ue8 quello di riuscire ad identificare dei trattamenti terapeutici efficaci che vadano a sostituire le cure palliative, le uniche disponibili oggi per questa malattia. Esistono diverse forme di demenza e, tra esse, la Malattia di Alzheimer (MA) \ue8 la pi\uf9 frequente, seguita dalla Demenza Vascolare (DV) e dalla Degenerazione Frontotemporale Lobare (FTLD), la quale, rappresenta il sottotipo pi\uf9 comune dopo la MA negli individui al di sotto dei 65 anni. Tutti e tre questi disordini possono presentare una componente genetica e, malgrado i notevoli investimenti e progressi degli ultimi anni per chiarirne le basi molecolari, la conoscenza dei meccanismi patologici che li determinano \ue8 ancora limitata. Tre forme di demenza, che possono avere una componente genetica, sono state incluse in questo studio: la Malattia di Alzheimer, la Degenerazione Frontotemporale Lobare e l'Arteriopatia Cerebrale Autosomica Dominante con Infarti Sottocorticali e Leucoencefalopatia (CADASIL), quest'ultima rappresentante un sottotipo di DV. I principali obiettivi di questo studio hanno compreso l'analisi mutazionale dei geni associati a queste malattie con il duplice intento di definirne le frequenze mutazionali in una casistica italiana e di valutare l'esistenza di eventuali correlazioni genotipo-fenotipo. Lo scopo finale era quello di definire degli algoritmi diagnostici utili ai medici specialisti nella selezione dei pazienti da sottoporre ad indagine genetica. Sono state allestite due coorti: una comprendente pazienti con diagnosi di MA, FTLD o disordini correlati, e l'altra, pazienti con CADASIL. L'analisi mutazionale \ue8 stata eseguita mediante due principali approcci: la Cromatografia Liquida Denaturante (DHPLC) ed il sequenziamento diretto. Sono stati analizzati i seguenti geni: proteina Precursore dell'Amiloide (APP), Presenilina1 (PSEN1) e Presenilina2 (PSEN2) per la Malattia di Alzheimer; Progranulina (PGRN), Proteina Tau Associata ai Microtubuli (MAPT), Proteina Contenente Valosina (VCP), Proteina di Modificazione della Cromatina 2B (CHMP2B), Proteina Legante TAR DNA (TARDBP) per la Degenerazione Frontotemporale Lobare ed il gene NOTCH3 per il CADASIL. L'analisi mutazionale dei geni associati a MA e FTLD ha portato all'identificazione di 8 mutazioni: 3 nuove variazioni, PSEN1 p.Ile437Asn, PSEN2 p.Thr18Met e PGRN p.His400ThrfsX12 e, cinque sostituzioni gi\ue0 descritte, PSEN2 p.Arg71Trp e p.Met174Val, PGRN p.Phe86SerfsX170 e p.Thr272SerfsX10 e MAPT c.IVS10+16C>T. I risultati delle analisi molecolari e delle simulazioni al computer depongono a favore di una natura patogenetica per le variazioni dei geni Presenilina, malgrado il ruolo di alcune di esse sia dibattuto in letteratura. Le sostituzioni identificate nei geni PGRN e MAPT sono mutazioni il cui meccanismo patologico \ue8 gi\ue0 stato descritto. Il fenotipo clinico riscontrato nei pazienti con le mutazioni identificate non era univoco e, specialmente le mutazioni nella PGRN erano associate ad una notevole variabilit\ue0 delle manifestazioni cliniche, anche all'interno di una stessa famiglia. Inoltre, l'identificazione di una famiglia con diagnosi di FTD-MND in linkage al cromosoma 9 sottolinea ulteriormente l'eterogeneit\ue0 genetica delle FTLD. L'analisi mutazionale del gene NOTCH3 ha portato all'identificazione di 21 diverse mutazioni, incluse 7 nuove variazioni, distribuite non uniformemente lungo il gene. Si \ue8 osservata una clusterizzazione delle mutazioni in aree geografiche diverse: la maggior parte identificate in pazienti provenienti dal nord-est Italia, alcune nel nord-ovest ed altre nell'Italia centrale. Nell'ipotesi che questa regionalizzazione rispecchiasse un effetto fondatore, si sono studiati gli aplotipi associati a queste mutazioni: i risultati, sebbene compatibili, non hanno permesso di confermare l'effetto fondatore in quanto la maggior parte di queste variazioni cadeva nell'aplotipo pi\uf9 frequente. I risultati di questo studio, complessivamente sono stati utili per definire degli algoritmi diagnostici che potrebbero aiutare i clinici ad identificare i pazienti, suggestivi di una base molecolare, da sottoporre ad analisi genetica.Dementia is a clinical syndrome associated with progressive deterioration of intellectual functions including loss of memory, difficulties with language, simple calculations, planning and judgment, and motor skills with eventually loss of autonomy. Currently there are an estimated 36 million people worldwide with dementia and this figure is set to increase to more than 115 million people by 2050. Given the prevalence of dementia and the associated significant financial and human costs, in recent years there has been a huge burst of studies aimed to identify the causes of this disorder and its underlain pathological mechanisms, in order to define therapeutic treatments to replace the nowadays available palliative cares. Among different subtypes of dementia, Alzheimer's Disease (AD) is the most frequent form, followed by Vascular Dementia and Frontotemporal Lobar Degeneration (FTLD) which represents the second most common form in people younger than 65 years. All three of these diseases may have a genetic component and, despite considerable progress and efforts made in recent years to clarify their molecular basis, little is known about the pathological mechanisms determining these diseases. Three forms of dementia, which may have a genetic component, were included in this study: Alzheimer's Disease, Frontotemporal Lobar Degeneration and Cerebral Autosomal Dominant Arteriopathy with Subcortical Infarcts and Leuokoencephalopathy (CADASIL) which is a subtype of vascular dementia. The main objectives of this study included mutational analysis of the genes associated with these diseases with the dual purpose of defining their mutational frequencies in an Italian series and to evaluate the presence of possible genotype-phenotype correlations. The secondary purpose is to be able to draw diagnostic algorithms useful to clinicians in selecting which patients submit to genetic testing. Two clinical cohorts were set up; one consisting in patients with diagnosis of AD, FTLD or related disorders, and the other made up of patients with CADASIL. The mutational analysis was performed by two main techniques: Denaturing High Performance Liquid Chromatography and direct sequencing. The analyzed genes encompassed: Amyloid Precursor Protein (APP), Presenilin1 (PSEN1) and Presenilin2 (PSEN2) for Alzheimer's Disease; Progranulin (PGRN), Microtubule Associated Protein Tau (MAPT), Valosin Containing Protein (VCP), Charged Multivesicular Body Protein 2B (CHMP2B), TAR DNA Binding Protein (TARDBP) for Frontotemporal Lobar Degeneration and NOTCH3 for CADASIL. The mutational analysis of AD- and FTLD-associated genes led to the identification of 8 mutations: three novel variations, PSEN1 p.Ile437Asn, PSEN2 p.Thr18Met and PGRN p.His400ThrfsX12 and five already described substitutions, PSEN2 p.Arg71Trp and p.Met174Val, PGRN p.Phe86SerfsX170 and p.Thr272SerfsX10 and MAPT c.IVS10+16C>T. The molecular data and in silico analyses performed in this study argue in favour of pathogenetic nature for Presenilins variations even though the role of some of them is debated in literature. The substitution identified in PGRN and MAPT are mutations whose pathogenic mechanism has already been described. The clinical phenotype associated to identified mutations was not unique and, especially mutations in the PGRN gene showed a marked variability in clinical presentations, even within the same family. Furthermore, the identification of a FTD-MND family linked to a locus on chromosome 9 further emphasizes the genetic heterogeneity of FTLD. The mutational screening of NOTCH3 gene led to the identification of 21 different mutations, including 7 novel variations, distributed unevenly along the gene. A geographical clustering was observed with mutations identified only in patients living in North-East Italy, a few in North-West and other in Central Italy. Haplotype analysis was performed to assess a possible founder effected underlying this regionalization but, although consistent, it was not confirmed as the majority of mutations was associated with the most common haplotype. The results of this study together were useful to define diagnostic algorithms that could help clinicians to identify patients suggestive of a molecular basis of disease to address to genetic testing

Metabolomics as a Powerful Tool for Molecular Quality Assessment of the Fish Sparus aurata

The molecular profiles of perchloric acid solutions extracted from the flesh of Sparus aurata fish specimens, produced according to different aquaculture systems, have been investigated. The 1H-NMR spectra of aqueous extracts are indicative of differences in the metabolite content of fish reared under different conditions that are already distinguishable at their capture, and substantially maintain the same differences in their molecular profiles after sixteen days of storage under ice. The fish metabolic profiles are studied by top-down chemometric analysis. The results of this exploratory investigation show that the fish metabolome accurately reflects the rearing conditions. The level of many metabolites co-vary with the rearing conditions and a few metabolites are quantified including glycogen (stress indicator), histidine, alanine and glycine which all display significant changes dependent on the aquaculture system and on the storage times

High diagnostic accuracy of RT-QuIC assay in a prospective study of patients with suspected sCJD

The early and accurate in vivo diagnosis of sporadic Creutzfeldt-Jakob disease (sCJD) is essential in order to differentiate CJD from treatable rapidly progressive dementias. Diagnostic investigations supportive of clinical CJD diagnosis include magnetic resonance imaging (MRI), electroencephalogram (EEG), 14-3-3 protein detection, and/or real-time quaking-induced conversion (RT-QuIC) assay positivity in the cerebrospinal fluid (CSF) or in other tissues. The total CSF tau protein concentration has also been used in a clinical setting for improving the CJD diagnostic sensitivity and specificity. We analyzed 182 CSF samples and 42 olfactory mucosa (OM) brushings from patients suspected of having sCJD with rapidly progressive dementia (RPD), in order to determine the diagnostic accuracy of 14-3-3, the total tau protein, and the RT-QuIC assay. A probable and definite sCJD diagnosis was assessed in 102 patients. The RT-QuIC assay on the CSF samples showed a 100% specificity and a 96% sensitivity, significantly higher compared with 14-3-3 (84% sensitivity and 46% specificity) and tau (85% sensitivity and 70% specificity); however, the combination of RT-QuIC testing of the CSF and OM samples resulted in 100% sensitivity and specificity, proving a significantly higher accuracy of RT-QuIC compared with the surrogate biomarkers in the diagnostic setting of patients with RPD. Moreover, we showed that CSF blood contamination or high protein levels might interfere with RT-QuIC seeding. In conclusion, we provided further evidence that the inclusion of an RT-QuIC assay of the CSF and OM in the diagnostic criteria for sCJD has radically changed the clinical approach towards the diagnosis

Handbook of remote working for social innovators

Sem resumo disponível.publishe

Metabolomics as a Powerful Tool for Molecular Quality Assessment of the Fish Sparus aurata

The molecular profiles of perchloric acid solutions extracted from the flesh of Sparus aurata fish specimens, produced according to different aquaculture systems, have been investigated. The 1H-NMR spectra of aqueous extracts are indicative of differences in the metabolite content of fish reared under different conditions that are already distinguishable at their capture, and substantially maintain the same differences in their molecular profiles after sixteen days of storage under ice. The fish metabolic profiles are studied by top-down chemometric analysis. The results of this exploratory investigation show that the fish metabolome accurately reflects the rearing conditions. The level of many metabolites co-vary with the rearing conditions and a few metabolites are quantified including glycogen (stress indicator), histidine, alanine and glycine which all display significant changes dependent on the aquaculture system and on the storage times

Handbook of remote working for social innovators

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Rare mutations in SQSTM1 modify susceptibility to frontotemporal lobar degeneration

Mutations in the gene coding for Sequestosome 1 (SQSTM1) have been genetically associated with amyotrophic lateral sclerosis (ALS) and Paget disease of bone. In the present study, we analyzed the SQSTM1 coding sequence for mutations in an extended cohort of 1,808 patients with frontotemporal lobar degeneration (FTLD), ascertained within the European Early-Onset Dementia consortium. As control dataset, we sequenced 1,625 European control individuals and analyzed whole-exome sequence data of 2,274 German individuals (total n = 3,899). Association of rare SQSTM1 mutations was calculated in a meta-analysis of 4,332 FTLD and 10,240 control alleles. We identified 25 coding variants in FTLD patients of which 10 have not been described. Fifteen mutations were absent in the control individuals (carrier frequency < 0.00026) whilst the others were rare in both patients and control individuals. When pooling all variants with a minor allele frequency < 0.01, an overall frequency of 3.2 % was calculated in patients. Rare variant association analysis between patients and controls showed no difference over the whole protein, but suggested that rare mutations clustering in the UBA domain of SQSTM1 may influence disease susceptibility by doubling the risk for FTLD (RR = 2.18 [95 % CI 1.24-3.85]; corrected p value = 0.042). Detailed histopathology demonstrated that mutations in SQSTM1 associate with widespread neuronal and glial phospho-TDP-43 pathology. With this study, we provide further evidence for a putative role of rare mutations in SQSTM1 in the genetic etiology of FTLD and showed that, comparable to other FTLD/ALS genes, SQSTM1 mutations are associated with TDP-43 pathology

SINEUP non-coding RNAs rescue defective frataxin expression and activity in a cellular model of Friedreich's Ataxia

Friedreich's ataxia (FRDA) is an untreatable disorder with neuro- and cardio-degenerative progression. This monogenic disease is caused by the hyper-expansion of naturally occurring GAA repeats in the first intron of the FXN gene, encoding for frataxin, a protein implicated in the biogenesis of iron-sulfur clusters. As the genetic defect interferes with FXN transcription, FRDA patients express a normal frataxin protein but at insufficient levels. Thus, current therapeutic strategies are mostly aimed to restore physiological FXN expression. We have previously described SINEUPs, natural and synthetic antisense long non-coding RNAs, which promote translation of partially overlapping mRNAs through the activity of an embedded SINEB2 domain. Here, by in vitro screening, we have identified a number of SINEUPs targeting human FXN mRNA and capable to up-regulate frataxin protein to physiological amounts acting at the post-transcriptional level. Furthermore, FXN-specific SINEUPs promote the recovery of disease-associated mitochondrial aconitase defects in FRDA-derived cells. In summary, we provide evidence that SINEUPs may be the first gene-specific therapeutic approach to activate FXN translation in FRDA and, more broadly, a novel scalable platform to develop new RNA-based therapies for haploinsufficient diseases