Enhanced genotypability for a more accurate variant calling in targeted resequencing

The analysis of Next-Generation Sequencing (NGS) data for the identification of DNA genetic variants presents several bioinformatics challenges. The main requirements of the analysis are the accuracy and the reproducibility of results, as their clinical interpretation may be influenced by many variables, from the sample processing to the adopted bioinformatics algorithms. Targeted resequencing, which aim is the enrichment of genomic regions to identify genetic variants possibly associated to clinical diseases, bases the quality of its data on the depth and uniformity of coverage, for the differentiation between true and false positives findings. Many variant callers have been developed to reach the best accuracy considering these metrics, but they can\u2019t work in regions of the genome where short reads cannot align uniquely (uncallable regions). The misalignment of reads on the reference genome can arise when reads are too short to overcome repetitious regions of the genome, causing the software to assign a low-quality score to the read pairs of the same fragment. A limitation of this process is that variant callers are not able to call variants in these regions, unless the quality of one of the two read mates could increase. Moreover, current metrics are not able to define with accuracy these regions, lacking in providing this information to the final customer. For this reason, a more accurate metric is needed to clearly report the uncallable genomic regions, with the prospect to improve the data analysis to possibly investigate them. This work aimed to improve the callability (genotypability) of the target regions for a more accurate data analysis and to provide a high-quality variant calling. Different experiments have been conducted to prove the relevance of genotypability for the evaluation of targeted resequencing performance. Firstly, this metric showed that increasing the depth of sequencing to rescue variants is not necessary at thresholds where genotypability reaches saturation (70X). To improve this metric and to evaluate the accuracy and reproducibility of results on different enrichment technologies for WES sample processing, the genotypability was evaluated on four exome platforms using three different DNA fragment lengths (short: ~200, medium: ~350, long: ~500 bp). Results showed that mapping quality could successfully increase on all platforms extending the fragment, hence increasing the distance between the read pairs. The genotypability of many genes, including several ones associated to a clinical phenotype, could strongly improve. Moreover, longer libraries increased uniformity of coverage for platforms that have not been completely optimized for short fragments, further improving their genotypability. Given the relevance of the quality of data derived, especially from the extension of the short fragments to the medium ones, a deeper investigation was performed to identify a potential threshold of fragment length above which the improvement in genotypability was significant. On the enrichment platform producing the higher enrichment uniformity (Twist), the fragments above 230 bp could obtain a meaningful improvement of genotypability (almost 1%) and a high uniformity of coverage of the target. Interestingly, the extension of the DNA fragment showed a greater influence on genotypability in respect on the solely uniformity of coverage. The enhancement of genotypability for a more accurate bioinformatics analysis of the target regions provided at limited costs (less sequencing) the investigation of regions of the genome previously defined as uncallable by current NGS methodologies

Genetic and functional characterization of disease associations explains comorbidity

Understanding relationships between diseases, such as comorbidities, has important socio-economic implications, ranging from clinical study design to health care planning. Most studies characterize disease comorbidity using shared genetic origins, ignoring pathway-based commonalities between diseases. In this study, we define the disease pathways using an interactome-based extension of known disease-genes and introduce several measures of functional overlap. The analysis reveals 206 significant links among 94 diseases, giving rise to a highly clustered disease association network. We observe that around 95% of the links in the disease network, though not identified by genetic overlap, are discovered by functional overlap. This disease network portraits rheumatoid arthritis, asthma, atherosclerosis, pulmonary diseases and Crohn's disease as hubs and thus pointing to common inflammatory processes underlying disease pathophysiology. We identify several described associations such as the inverse comorbidity relationship between Alzheimer's disease and neoplasms. Furthermore, we investigate the disruptions in protein interactions by mapping mutations onto the domains involved in the interaction, suggesting hypotheses on the causal link between diseases. Finally, we provide several proof-of-principle examples in which we model the effect of the mutation and the change of the association strength, which could explain the observed comorbidity between diseases caused by the same genetic alterations

Exosomes from Plasma of Neuroblastoma Patients Contain Doublestranded DNA Reflecting the Mutational Status of Parental Tumor Cells

Neuroblastoma (NB) is an aggressive infancy tumor, leading cause of death among preschool age diseases. Here we focused on characterization of exosomal DNA (exo-DNA) isolated from plasma cell-derived exosomes of neuroblastoma patients, and its potential use for detection of somatic mutations present in the parental tumor cells. Exosomes are small extracellular membrane vesicles secreted by most cells, playing an important role in intercellular communications. Using an enzymatic method, we provided evidence for the presence of double-stranded DNA in the NB exosomes. Moreover, by whole exome sequencing, we demonstrated that NB exo-DNA represents the entire exome and that it carries tumor-specific genetic mutations, including those occurring on known oncogenes and tumor suppressor genes in neuroblastoma (ALK, CHD5, SHANK2, PHOX2B, TERT, FGFR1, and BRAF). NB exo-DNA can be useful to identify variants responsible for acquired resistance, such as mutations of ALK, TP53, and RAS/MAPK genes that appear in relapsed patients. The possibility to isolate and to enrich NB derived exosomes from plasma using surface markers, and the quick and easy extraction of exo-DNA, gives this methodology a translational potential in the clinic. Exo-DNA can be an attractive non-invasive biomarker for NB molecular diagnostic, especially when tissue biopsy cannot be easily available

Physical Activity and Nutrition INfluences in Ageing: Current Findings from the PANINI Project

Background: The ageing of the population is a global challenge and the period of life spent in good health, although increasing, is not keeping pace with lifespan. Consequently, understanding the important factors that contribute to healthy ageing and validating interventions and influencing policy to promote healthy ageing are vital research priorities. Method: The PANINI project is a collaboration of 20 partners across Europe examining the influence of physical activity and nutrition in ageing. Methods utilised encompass the biological to the social, from genetics to the influence of social context. For example, epigenetic, immunological, and psychological assessments, and nutritional and sports science-based interventions have been used among older adults, as well as mathematical modelling and epidemiology. The projects are multi-disciplinary and examine health outcomes in ageing from a range of perspectives. Results: The results discussed here are those emerging thus far in PANINI from 11 distinct programmes of research within PANINI as well as projects cross-cutting the network. New approaches, and the latest results are discussed. Conclusions: The PANINI project has been addressing the impact of physical activity and nutrition on healthy ageing from diverse but interlinked perspectives. It emphasises the importance of using standardized measures and the advantages of combining data to compare biomarkers and interventions across different settings and typologies of older adults. As the projects conclude, the current results and final data will form part of a shared dataset, which will be made open access for other researchers into ageing processes.On behalf of the PANINI Consortiu

An isoform of the giant protein titin is a master regulator of human T lymphocyte trafficking

Response to multiple microenvironmental cues and resilience to mechanical stress are essential features of trafficking leukocytes. Here, we describe unexpected role of titin (TTN), the largest protein encoded by the human genome, in the regulation of mechanisms of lymphocyte trafficking. Human T and B lymphocytes express five TTN isoforms, exhibiting cell-specific expression, distinct localization to plasma membrane microdomains, and different distribution to cytosolic versus nuclear compartments. In T lymphocytes, the LTTN1 isoform governs the morphogenesis of plasma membrane microvilli independently of ERM protein phosphorylation status, thus allowing selectin-mediated capturing and rolling adhesions. Likewise, LTTN1 controls chemokine-triggered integrin activation. Accordingly, LTTN1 mediates rho and rap small GTPases activation, but not actin polymerization. In contrast, chemotaxis is facilitated by LTTN1 degradation. Finally, LTTN1 controls resilience to passive cell deformation and ensures T lymphocyte survival in the blood stream. LTTN1 is, thus, a critical and versatile housekeeping regulator of T lymphocyte trafficking

SOX2: Not always eye malformations. Severe genital but no major ocular anomalies in a female patient with the recurrent c.70del20 variant

SOX2 variants have been identified in multiple patients with severe ocular anomalies and pituitary dysfunction, in addition to various systemic features. We investigated a 26-year-old female patient suffering from spastic paraparesis, hypoplasia of corpus callosum, hypogonadotropic hypogonadism (HH) and intellectual disability, who was monitored for over 20 years, allowing a detailed genotype-phenotype correlation along time. Whole exome sequencing on the patient and her relatives identified a de novo SOX2 c.70del20 variant, which has been frequently reported in individuals with SOX2-related anophthalmia. Importantly, our patient lacked major ocular phenotype but showed vaginal agenesis, a feature never reported before. Although the involvement of male urogenital tract (cryptorchidism, hypospadias, small penis), is a well known consequence of SOX2 variants, their effect on the female genitalia has never been properly addressed, even considering the paradoxical female excess of SOX2 cases in the literature. Our findings emphasize the importance of testing for SOX2 variants in individuals with HH and genital anomalies even though anophthalmia or microphthalmia are not observed. Moreover, our case strengthens the role of SOX2 as a master regulator of female gonadal differentiation, as widely demonstrated for other SOX genes related to 46, XX sex reversal, such as SOX3 and SOX9

Fenomeni di diffusione delle notizie in siti di microblogging: modelli di analisi e studio di caso.

L'informazione è alla base della conoscenza umana. Senza, non si potrebbe sapere nulla di ciò che esiste, di ciò che è stato o di quello che potrebbe accadere. Ogni giorno si assimilano moltissime informazioni, che vengono registrate nella propria memoria per essere riutilizzate all'occorrenza. Ne esistono di vari generi, ma il loro insieme va a formare quella che è la cultura, educazione, tradizione e storia dell'individuo. Per questo motivo è importante la loro diffusione e salvaguardia, impedendone la perdita che costerebbe la dipartita di una parte di sé, del proprio passato o del proprio futuro. Al giorno d'oggi le informazioni possono essere acquisite tramite persone, libri, riviste, giornali, la televisione, il Web. I canali di trasmissione sono molti, alcuni più efficaci di altri. Tra questi, internet è diventato un potente strumento di comunicazione, il quale consente l'interazione tra chi naviga nel Web (ossia gli utenti) e una partecipazione attiva alla diffusione di informazioni. Nello specifico, esistono siti (chiamati di microblogging) in cui sono gli stessi utenti a decidere se un'informazione possa essere o meno inserita nella propria pagina personale. In questo caso, si è di fronte a una nuova "gestione dell'informazione", che può variare da utente a utente e può defluire in catene di propagazione (percorsi che compiono i dati e le notizie tra i navigatori del Web) dai risvolti spesso incerti. Ma esiste un modello che possa spiegare l'avanzata delle informazioni tra gli utenti? Se fosse possibile capirne la dinamica, si potrebbe venire a conoscenza di quali sono le informazioni più soggette a propagazione, gli utenti che più ne influenzano i percorsi, quante persone ne vengono a conoscenza o il tempo per cui resta attiva un'informazione, descrivendone una sorta di ciclo di vita. E' possibile nel mondo reale trovare delle caratteristiche ricorrenti in queste propagazioni, in modo da poter sviluppare un metodo universale per acquisirne e analizzarne le dinamiche? I siti di microblogging non seguono regole precise, perciò si va incontro a un insieme apparentemente casuale di informazioni che necessitano una chiave di lettura. Quest'ultima è proprio quella che si è cercata, con la speranza di poter sfruttare i risultati ottenuti nell'ipotesi di una futura gestione dell'informazione più consapevole. L'obiettivo della tesi è quello di identificare un modello che mostri con chiarezza quali sono i passaggi da affrontare nella ricerca di una logica di fondo nella gestione delle informazioni in rete

#rescatemineros: global media events in the microblogging age

Contemporary media events are experienced within the complex scenario of convergence media. Broadcasting channels and traditional mass media coexist with online digital news channels and with countless social media services. This paper describes how a global media event (the rescue operation of the 33 Chilean miners trapped underground for 69 days between August and October 2010) can be observed through the communication occurred inside Twitter, the largest microblogging platform. The article proposes a data-driven methodology that, starting from a large data acquisition procedure, is used to investigate: a) what communication patterns emerged within the microblogging sites; and b) how relevant information is propagated through the microblogging network of users