1,721,002 research outputs found
EXOME SEQUENCING APPROACH TO IDENTIFY CAUSATIVE GENES FOR AMYOTROPHIC LATERAL SCLEROSIS
Full text linkIntroduzione: La Sclerosi laterale amiotrofica (SLA) è una malattia neurodegenerativa progressiva e fatale caratterizzata dalla perdita selettiva dei motoneuroni nella corteccia cerebrale, nel tronco cerebrale e midollo spinale. La maggior parte dei casi è costituita da forme sporadiche, mentre solo il 5-10% dei casi è rappresentato da forme familiari, causate da geni con modalità di trasmissione mendeliana, generalmente autosomica dominante. Sono stati identificati più di 20 geni causativi delle forme familiari, che hanno contribuito a comprendere meglio i meccanismi patogenetici coinvolti e sottolineano la grande eterogeneità genetica della malattia. Nonostante i numerosi progressi raggiunti, in circa il 40% dei casi familiari la causa genetica non è stata ancora identificata, mentre la componente genetica delle forme sporadiche è in gran parte sconosciuta.
L’ applicazione delle tecniche di sequenziamento di nuova generazione ed in particolare il sequenziamento della porzione codificante del genoma o esoma rappresenta un approccio innovativo e promettente per gli studi genetici sulla SLA. Lo scopo del presente progetto di ricerca di Dottorato è stato quello di identificare nuovi geni associati alle forme familiari e sporadiche di SLA mediante sequenziamento dell’esoma come metodo alternativo per superare i limiti delle tecniche genetiche tradizionali.
Metodi: Sono state utilizzate tre diverse strategie per l’identificazione di geni causativi: I) sequenziamento dell’esoma in combinazione con analisi di linkage in due grandi famiglie SLA a trasmissione dominante; II) analisi per varianti rare degli esomi di 363 casi familiari singoli; III) sequenziamento dell’esoma in 32 casi di SLA sporadica e dei loro genitori non affetti (approccio dei trios).
Risultati: I) Attraverso l’approccio combinato di sequenziamento dell’esoma ed analisi di linkage, abbiamo identificato il gene PFN1 (profilina-1), codificante per una proteina implicata nella regolazione dell’actina, come nuovo gene causativo di SLA. Mutazioni a carico del gene PFN1 sono state osservate nel 2,6% dei pazienti SLA familiari e gli studi funzionali condotti sui mutanti hanno dimostrato una maggiore tendenza all’aggregazione, una riduzione del legame all’actina ed un effetto inibitorio sulla crescita assonale.
II) L’analisi delle varianti rare tra casi e controlli, applicata su un totale di 12.495 geni, ha portato all’identificazione di TUBA4A (codificante per lalfa-tubulina 4a) come gene candidato caratterizzato da un eccesso significativo di varianti rare potenzialmente dannose nei 363 casi familiari analizzati. L’analisi funzionale ha dimostrato per i mutanti di TUBA4A una capacità ridotta di dimerizzazione con la beta-tubulina in vitro ed un’alterata incorporazione nei microtubuli in vivo. Inoltre, il mutante tronco TUBA4A p.W407X ha mostrato una maggiore tendenza all’aggregazione.
III) Infine, analizzando l’esoma di 32 pazienti con SLA sporadica e dei loro genitori non affetti, abbiamo identificato 25 mutazioni de novo in 16 dei 32 trios analizzati, con un tasso di mutazioni de novo pari a 0,78. Non sono stati identificati geni con molteplici mutazioni de novo nei trios sequenziati, ma le analisi bioinformatiche hanno mostrato possibili connessioni tra i geni candidati e la classificazione funzionale ha rilevato che le mutazioni de novo sono principalmente a carico di geni codificanti per trasportatori o per proteine con attività regolatoria sulle GTPasi.
Conclusioni: I risultati ottenuti hanno dimostrato che il sequenziamento dell’esoma, applicato con specifiche strategie di studio e di analisi, è un approccio efficace per l’identificazione di nuovi geni causativi nella SLA. La scoperta dei due geni PFN1 e TUBA4A, codificanti per proteine coinvolte nel processo di polimerizzazione dell’actina e dei microtubuli, fornisce ulteriori evidenze a supporto del coinvolgimento del citoscheletro nella patogenesi della SLA.Introduction: Amyotrophic lateral sclerosis (ALS) is an adult-onset neurodegenerative disorder caused by the loss of motor neurons in the cerebral cortex, brainstem and spinal cord. ALS occurs prevalently as sporadic forms (SALS), but a small proportion of cases (5-10%) displays a positive family history (FALS), generally with an autosomal dominant pattern of inheritance. To date, more than 20 causative genes have been identified in FALS, providing fundamental insights into the pathogenic mechanisms and underlying the great genetic heterogeneity of the disease. Despite these numerous advances, the genetic basis of nearly 40% of FALS remains to be identified, while the genetic component of SALS is largely unknown. A powerful and innovative tool for genetic studies in ALS is represented by next-generation sequencing and in particular by the targeted sequencing of the coding part of the genome or exome.
Aim of this research project was to identify novel genes associated to FALS and SALS by applying complementary approaches all based on exome sequencing, which overcomes the limitations of traditional genetic strategies.
Methods: Three different disease gene identification strategies were applied: I) exome sequencing associated to linkage analysis in two large ALS dominant pedigrees; II) exome-wide rare variant burden analysis on 363 unrelated index FALS cases; III) exome sequencing of 32 SALS and their unaffected parents (trio-design).
Results: I) By performing exome-sequencing in combination with linkage analysis, we identified PFN1 (profilin-1), encoding for a protein regulating actin dynamics, as a novel ALS-causative gene. Mutations in PFN1 were observed in ~2.6% of FALS and functional studies demonstrated aggregation propensity, reduction of actin binding ability and axonal outgrowth inhibition of mutant PFN1 proteins.
II) As a result of the unbiased case-control rare variant analysis, applied on a total 12.495 genes, we identified TUBA4A (Tubulin, Alpha 4a) as candidate gene showing a statistically significant excess of rare damaging variants in 363 index FALS cases sequenced. Functional analysis revealed that ALS-related mutants were defective in forming alpha/beta tubulin dimers in vitro and in incorporating into microtubules in vivo. In addition, the truncated mutant TUBA4A p.W407X showed aggregation propensities.
III) By sequencing the exomes of 32 SALS patients and their unaffected parents, we identified 25 de novo mutations (DNMs) in 16 of 32 trios, with an overall DNM rate of 0.78. Although we did not find recurrently mutated genes in our ALS trios, bionformatic analysis showed potential inter-connections between the candidate genes. Functional classification revealed that DNMs are enriched in genes encoding for proteins involved in transport and in GTPase regulatory activity.
Conclusions: Our findings indicate that exome-sequencing, combined with different strategies for study design and data analysis, is an effective and successful approach for the identification of novel ALS causative genes. The identification of PFN1 and TUBA4A genes, encoding for proteins implicated, respectively, in actin polymerization and microtubule formation, further supports a major role of cytoskeletal defects in ALS pathogenesis
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Variations on the Author
Full text link“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Appropriate Similarity Measures for Author Cocitation Analysis
Full text linkWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
Apoptosis induced by proteasome inhibition in human myoblast cultures
No full textDysfunction of the ubiquitin-proteasome system has recently been implicated in the pathogenesis of some untreatable myodegenerative diseases characterized by the formation of ubiquitinated inclusions in skeletal muscles. We have developed an in vitro model of proteasomal dysfunction by applying inhibitors of the proteasome to primary adult human skeletal muscle cultures. Our data show that proteasome inhibition causes both cytoplasmic accumulation of ubiquitinated inclusions and apoptotic death, the latter through accumulation of active caspase-3
Loss of Parkin Protein causes abnormal intracellular calcium levels upon glutamatergic stimulation in neuronal cells
No full textRNA-binding proteins and RNA metabolism : a new scenario in the pathogenesis of Amyotrophic Lateral Sclerosis
No full textSeveral RNA-processing genes have been implicated in the pathogenesis of Amyotrophic lateral sclerosis (ALS). In particular, causative mutations in the genes encoding for two DNA/RNA binding proteins, TAR DNA binding protein-43 (TDP-43) and fused in sarcoma/translocated in liposarcoma (FUS/TLS), were recently identified in ALS patients. These genetic findings and the presence of abnormal aggregates of these two RNA-binding proteins in ALS affected tissues suggest that molecular mechanisms regulating RNA metabolism are implicated in ALS pathogenesis through common pathways. In this review similarities and differences between TDP-43 and FUS/TLS proteins and their activities in physiological and pathological conditions will be discussed
Dispelling the Myths Behind First-author Citation Counts
Full text linkWe conducted a full-scale evaluative citation analysis study of scholars in the XML research field to explore just how different from each other author rankings resulting from different citation counting methods actually are, and to demonstrate the capability of emerging data and tools on the Web in supporting more realistic citation counting methods. Our results contest some common arguments for the continued
use of first-author citation counts in the evaluation of scholars, such as high correlations between author rankings by first-author citation counts and other citation
counting methods, and high costs of using more realistic citation counting methods that are not well-supported by the ISI databases. It is argued that increasingly available digital full text research papers make it possible for citation analysis studies to go beyond what the ISI databases have directly supported and to employ more
sophisticated methods
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