C6orf10 low-frequency and rare variants in italian multiple sclerosis patients

In light of the complex nature of multiple sclerosis (MS) and the recently estimated contribution of low-frequency variants into disease, decoding its genetic risk components requires novel variant prioritization strategies. We selected, by reviewing MS Genome Wide Association Studies (GWAS), 107 candidate loci marked by intragenic single nucleotide polymorphisms (SNPs) with a remarkable association (p-value <= 5 x 10(-6)). A whole exome sequencing (WES)-based pilot study of SNPs with minor allele frequency (MAF) <= 0.04, conducted in three Italian families, revealed 15 exonic low-frequency SNPs with affected parent-child transmission. These variants were detected in 65/120 Italian unrelated MS patients, also in combination (22 patients). Compared with databases (controls gnomAD, dbSNP150, ExAC, Tuscany-1000 Genome), the allelic frequencies of C6orf10 rs 16870005 and IL2RA rs12722600 were significantly higher (i.e., controls gnomAD, p = 9.89 x 10(-7) and p < 1 x 10(-20)). TET2 rs61744960 and TRAF3 rs138943371 frequencies were also significantly higher, except in Tuscany-1000 Genome. Interestingly, the association of C6orf10 rs16870005 (Ala431Thr) with MS did not depend on its linkage disequilibrium with the HLA-DRB1 locus. Sequencing in the MS cohort of the C6orf10 3' region revealed 14 rare mutations (10 not previously reported). Four variants were null, and significantly more frequent than in the databases. Further, the C6orf10 rare variants were observed in combinations, both intra-locus and with other low-frequency SNPs. The C6orf10 Ser389Xfr was found homozygous in a patient with early onset of the MS. Taking into account the potentially functional impact of the identified exonic variants, their expression in combination at the protein level could provide functional insights in the heterogeneous pathogenetic mechanisms contributing to MS.In light of the complex nature of multiple sclerosis (MS) and the recently estimated contribution of low-frequency variants into disease, decoding its genetic risk components requires novel variant prioritization strategies. We selected, by reviewing MS Genome Wide Association Studies (GWAS), 107 candidate loci marked by intragenic single nucleotide polymorphisms (SNPs) with a remarkable association (p-value ≤ 5 × 10−6). A whole exome sequencing (WES)-based pilot study of SNPs with minor allele frequency (MAF) ≤ 0.04, conducted in three Italian families, revealed 15 exonic low-frequency SNPs with affected parent-child transmission. These variants were detected in 65/120 Italian unrelated MS patients, also in combination (22 patients). Compared with databases (controls gnomAD, dbSNP150, ExAC, Tuscany-1000 Genome), the allelic frequencies of C6orf10 rs16870005 and IL2RA rs12722600 were significantly higher (i.e., controls gnomAD, p = 9.89 × 10−7 and p < 1 × 10−20). TET2 rs61744960 and TRAF3 rs138943371 frequencies were also significantly higher, except in Tuscany-1000 Genome. Interestingly, the association of C6orf10 rs16870005 (Ala431Thr) with MS did not depend on its linkage disequilibrium with the HLA-DRB1 locus. Sequencing in the MS cohort of the C6orf10 3′ region revealed 14 rare mutations (10 not previously reported). Four variants were null, and significantly more frequent than in the databases. Further, the C6orf10 rare variants were observed in combinations, both intra-locus and with other low-frequency SNPs. The C6orf10 Ser389Xfr was found homozygous in a patient with early onset of the MS. Taking into account the potentially functional impact of the identified exonic variants, their expression in combination at the protein level could provide functional insights in the heterogeneous pathogenetic mechanisms contributing to MS

Microplastic pollution in the Trieste Karst (Italy) protected habitats: preliminary analysis of cave and spring water sediments

Microplastic (MP) pollution in karst and subterranean areas is still poorly studied and research are generally focused on the water matrix. The Trieste Karst, Italy, is rich in peculiar karst habitats and species, including some endemics. To preserve these ecological heritages, different European, national and local laws are present. In this preliminary study we collected and investigated several sediment samples from aquatic environments in different protected habitats (three caves and a spring) of the Trieste Karst. Sediment samples were subjected to organic matter removal with 1:1 30% H2O2 solution and for each sample, three subsamples of 15g dried sediment were selected via coning and quartering. MPs were extracted from sediment via density separation and filtered. Particles on filters (5-0.1 mm) were counted and characterized by size, color and shape via visual identification under a microscope, with and without UV light, exploiting fluorescence given by additives added in many plastic materials. Finally, spectroscopic analyses were carried out on random particles on each filter. The concentration of MPs in cave water sediments varied from 911 to 2178 items/kg, instead, in the sediments of the spring it was of 889 items/kg. Fibre represented the most abundant shape (67.5%), followed by fragment (21.6%), bead (10%), and film (0.9%). Most MPs (86.4%) were smaller than 1 mm. The majority of the MPs were fluorescent under UV light (69.1%) and have 77.1% blue fluorescence, 8.1% red fluorescence, 6.1% green fluorescence and 8.7% other colors. Fluorescent particles were mainly transparent (63.8%), instead, in non-fluorescent ones predominated the black (56.2%) and brown (15.1%) MPs. Our results show the presence of MPs in all examined aquatic habitats, providing essential information for future research. The studied areas are adjacent to highways, roads and railways, therefore, most of the particles found in water sediment samples could come from surface pollution, transported by water and/or air. In addition, the waters of the sampling points are often stationary or poorly moved, therefore, there may be an accumulation of pollutants in the sediments. Vulnerable and troglobitic species hosted in these habitats could consume or assimilate MPs, which can irreversibly damage ecosystems and contaminate water resources too. Analyses on a greater number of aquatic surface and subterranean habitats should be done to better understand this kind of problem. Monitoring MPs pollution in karst areas should become a priority for the habitat conservation, the species protection and the water resources management

An exon-specific U1 small nuclear RNA (snRNA) strategy to correct splicing defects

A significant proportion of disease-causing mutations affect precursor-mRNA splicing, inducing skipping of the exon from the mature transcript. Using F9 exon 5, CFTR exon 12 and SMN2 exon 7 models, we characterized natural mutations associated to exon skipping in Haemophilia B, cystic fibrosis and spinal muscular atrophy (SMA), respectively, and the therapeutic splicing rescue by using U1 small nuclear RNA (snRNA). In minigene expression systems, loading of U1 snRNA by complementarity to the normal or mutated donor splice sites (5′ss) corrected the exon skipping caused by mutations at the polypyrimidine tract of the acceptor splice site, at the consensus 5′ss or at exonic regulatory elements. To improve specificity and reduce potential off-target effects, we developed U1 snRNA variants targeting non-conserved intronic sequences downstream of the 5′ss. For each gene system, we identified an exon-specific U1 snRNA (ExSpeU1) able to rescue splicing impaired by the different types of mutations. Through splicing-competent cDNA constructs, we demonstrated that the ExSpeU1-mediated splicing correction of several F9 mutations results in complete restoration of secreted functional factor IX levels. Furthermore, two ExSpeU1s for SMA improved SMN exon 7 splicing in the chromosomal context of normal cells. We propose ExSpeU1s as a novel therapeutic strategy to correct, in several human disorders, different types of splicing mutations associated with defective exon definition

Artificial intelligence for target prostate biopsy outcomes prediction the potential application of fuzzy logic

Background: In current precision prostate cancer (PCa) surgery era the identification of the best patients candidate for prostate biopsy still remains an open issue. The aim of this study was to evaluate if the prostate target biopsy (TB) outcomes could be predicted by using artificial intelligence approach based on a set of clinical pre-biopsy. Methods: Pre-biopsy characteristics in terms of PSA, PSA density, digital rectal examination (DRE), previous prostate biopsies, number of suspicious lesions at mp-MRI, lesion volume, lesion location, and Pi-Rads score were extracted from our prospectively maintained TB database from March 2014 to December 2019. Our approach is based on Fuzzy logic and associative rules mining, with the aim to predict TB outcomes. Results: A total of 1448 patients were included. Using the Frequent-Pattern growth algorithm we extracted 875 rules and used to build the fuzzy classifier. 963 subjects were classified whereas for the remaining 484 subjects were not classified since no rules matched with their input variables. Analyzing the classified subjects we obtained a specificity of 59.2% and sensitivity of 90.8% with a negative and the positive predictive values of 81.3% and 76.6%, respectively. In particular, focusing on ISUP ≥ 3 PCa, our model is able to correctly predict the biopsy outcomes in 98.1% of the cases. Conclusions: In this study we demonstrated that the possibility to look at several pre-biopsy variables simultaneously with artificial intelligence algorithms can improve the prediction of TB outcomes, outclassing the performance of PSA, its derivates and MRI alone

Machine-Learning-Based Tool to Predict Target Prostate Biopsy Outcomes: An Internal Validation Study

Abstract: The aim of this study is to present a personalized predictive model (PPM) with a machine learning (ML) system that is able to identify and classify patients with suspected prostate cancer (PCa) following mpMRI. We extracted all the patients who underwent fusion biopsy (FB) from March 2014 to December 2019, while patients from August 2020 to April 2021 were included as a validation set. The proposed system was based on the following four ML methods: a fuzzy inference system (FIS), the support vector machine (SVM), k-nearest neighbors (KNN), and self-organizing maps (SOMs). Then, a system based on fuzzy logic (FL) + SVM was compared with logistic regression (LR) and standard diagnostic tools. A total of 1448 patients were included in the training set, while 181 patients were included in the validation set. The area under the curve (AUC) of the proposed FIS + SVM model was comparable with the LR model but outperformed the other diagnostic tools. The FIS + SVM model demonstrated the best performance, in terms of negative predictive value (NPV), on the training set (78.5%); moreover, it outperformed the LR in terms of specificity (92.1% vs. 83%). Considering the validation set, our model outperformed the other methods in terms of NPV (60.7%), sensitivity (90.8%), and accuracy (69.1%). In conclusion, we successfully developed and validated a PPM tool using the FIS + SVM model to calculate the probability of PCa prior to a prostate FB, avoiding useless ones in 15% of the cases

MORFEO enters final design phase

MORFEO (Multi-conjugate adaptive Optics Relay For ELT Observations, formerly MAORY), the MCAO system for the ELT, will provide diffraction-limited optical quality to the large field camera MICADO. MORFEO has officially passed the Preliminary Design Review and it is entering the final design phase. We present the current status of the project, with a focus on the adaptive optics system aspects and expected milestones during the next project phase

Giustizia e letteratura II

The book explores and links different cultures, disciplines and perspectives, with a far more original and broad approach to the relations between “Justice” and “Literature” than more traditional works focused on “Law” and “Literature”. The many contributions from writers, literature and movie critics, psychologists, and criminal law practitioners and scholars, draw a complex and interdisciplinary path through primary texts of Italian and international literature, with the aim of prompting readers’ reflections about core issues related to law, crime, and responsibility. Through the analysis of masterpieces of literature, theatre and cinema, this book aims at stimulating dialogue and debate, as well as critical abilities and a deep-rooted sense of justice, amongst both law professionals and citizens at large. Literature and other forms of narration are presented here as a privileged key to approach long-standing questions about (amongst other) causes and consequences of crime; victimization and coping mechanisms; the role of criminal law and criminal proceedings; legalism and equity; law and ethics; the ‘time’ of justice; freedom, responsibility, culpability and forgiveness; rules, legality, socialization and culture; language and images as mediums for justice issues; the impact of prejudice and of existing balances of power on the application of the law; social and legal mechanisms of exclusion and inclusion; gender issues and legal systems; and so on. A whole section (Part V) is devoted to crimes against humanity and how the literary testimony may be understood both as a strategy to resist injustice and to seek justice, and as a way to prevent further horrors. Through this quest for justice in literature and arts, the volume proposes a wider cultural and research project which defies traditional formalistic and retributive approaches to criminal law, in order to open new perspectives for restorative and reintegrative strategies

La terapia genica nelle Malattie emorragiche e trombotiche

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An Altered Splicing Registry Explains the Differential ExSpeU1-Mediated Rescue of Splicing Mutations Causing Haemophilia A

The exon recognition and removal of introns (splicing) from pre-mRNA is a crucial step in the gene expression flow. The process is very complex and therefore susceptible to derangements. Not surprisingly, a significant and still underestimated proportion of disease-causing mutations affects splicing, with those occurring at the 5' splice site (5'ss) being the most severe ones. This led to the development of a correction approach based on variants of the spliceosomal U1snRNA, which has been proven on splicing mutations in several cellular and mouse models of human disease. Since the alternative splicing mechanisms are strictly related to the sequence context of the exon, we challenged the U1snRNA-mediated strategy in the singular model of the exon 5 of coagulation factor (F)VIII gene (F8) in which the authentic 5'ss is surrounded by various cryptic 5'ss. This scenario is further complicated in the presence of nucleotide changes associated with FVIII deficiency (Haemophilia A), which weaken the authentic 5'ss and create/strengthen cryptic 5'ss. We focused on the splicing mutations (c.602-32A &gt; G, c.602-10T &gt; G, c.602G &gt; A, c.655G &gt; A, c.667G &gt; A, c.669A &gt; G, c.669A &gt; T, c.670G &gt; T, c.670+1G &gt; T, c.670+1G &gt; A, c.670+2T &gt; G, c.670+5G &gt; A, and c.670+6T &gt; C) found in patients with severe to mild Haemophilia A. Minigenes expression studies demonstrated that all mutations occurring within the 5'ss, both intronic or exonic, lead to aberrant transcripts arising from the usage of two cryptic intronic 5'ss at positions c.670+64 and c.670+176. For most of them, the observed proportion of correct transcripts is in accordance with the coagulation phenotype of patients. In co-transfection experiments, we identified a U1snRNA variant targeting an intronic region downstream of the defective exon (Exon Specific U1snRNA, U1sh7) capable to re-direct usage of the proper 5'ss (similar to 80%) for several mutations. However, deep investigation of rescued transcripts from +1 and +2 variants revealed only the usage of adjacent cryptic 5'ss, leading to frameshifted transcript forms. These data demonstrate that a single ExSpeU1 can efficiently rescue different mutations in the F8 exon 5, and provide the first evidence of the applicability of the U1snRNA-based approach to Haemophilia A

Rescue of multiple Haemophilia A-causing mutations by a single ExSpeU1: the importance of the genomic context

Background: Therapies based on RNA splicing modulation are attracting interest for many disorders.. Variants of the spliceosomal U1snRNA have been successfully exploited to rescue defective exons in cellular and mouse models but no attempts have been done on Hemophilia A, the commonest coagulation disorder. Aims: To explore U1snRNA variants targeting intronic sequences downstreaom of the defective exon (exon-specific U1snRNA; ExSpeU1) to correct F8 exon 5 mutations leading to hemophilia A (HA). Methods: Expression of ExSpeU1s and F8 minigenes harboring the c.602-32A&gt;G, c.602-10T&gt;G, c.602G&gt;A, c.655G&gt;A, c.667G&gt;A, c.669A&gt;G, c.669A&gt;T, c.670G&gt;T, c.670+1G&gt;T, c.670+1G&gt;A, c.670+2T&gt;G, c.670+5G&gt;A and c.670+6T&gt;C mutations in HEK293T cells and evaluation of F8 mRNA splicing (RT-PCR). Results: Expression studies demonstrated that all mutations, both intronic and exonic, occurring within the 5’ splice site (5’ss) induced aberrant transcripts, with the usage of two cryptic intronic 5’ss at positions c.670+64 and c.670+176. Some changes were also associated to trace level of correct transcripts (~10%) and missense changes had no effect on splicing. In co-transfection experiments, we identified an ExSpeU1 (U1sh7), designed to minimize potential off-target effects, able to properly restore splicing. We showed in vitro that the ExSpeU1 is able to strengthen or restore (~80%) proper 5’ss usage for all splicing mutations, including changes at +1 and +2 positions of 5’ss, commonly considered not rescuable. However, deep investigation of rescued transcripts from +1 and +2 variants revealed the usage of adjacent subtle cryptic 5’ss, leading to frameshift. Conclusions: These data further support the therapeutic potential of the ExSpeU1 RNA, where a single therapeutic RNA can rescue multiple mutations. However, they suggest careful inspection of the genomic context and evaluation of transcripts to avoid over-interpretations