Allelic heterogeneity and abnormal vesicle recycling in PLAA-related neurodevelopmental disorders.

The human PLAA gene encodes Phospholipase-A2-Activating-Protein (PLAA) involved in trafficking of membrane proteins. Through its PUL domain (PLAP, Ufd3p, and Lub1p), PLAA interacts with p97/VCP modulating synaptic vesicles recycling. Although few families carrying biallelic PLAA variants were reported with progressive neurodegeneration, consequences of monoallelic PLAA variants have not been elucidated. Using exome or genome sequencing we identified PLAA de-novo missense variants, affecting conserved residues within the PUL domain, in children affected with neurodevelopmental disorders (NDDs), including psychomotor regression, intellectual disability (ID) and autism spectrum disorders (ASDs). Computational and in-vitro studies of the identified variants revealed abnormal chain arrangements at C-terminal and reduced PLAA-p97/VCP interaction, respectively. These findings expand both allelic and phenotypic heterogeneity associated to PLAA-related neurological disorders, highlighting perturbed vesicle recycling as a potential disease mechanism in NDDs due to genetic defects of PLAA.

Impaired Mitochondrial Function and Marrow Failure in Patients Carrying a Variant of the SRSF4 Gene.

Serine/arginine-rich splicing factors (SRSFs) are a family of proteins involved in RNA metabolism, including pre-mRNA constitutive and alternative splicing. The role of SRSF proteins in regulating mitochondrial activity has already been shown for SRSF6, but SRSF4 altered expression has never been reported as a cause of bone marrow failure. An 8-year-old patient admitted to the hematology unit because of leukopenia, lymphopenia, and neutropenia showed a missense variant of unknown significance of the SRSF4 gene (p.R235W) found via whole genome sequencing analysis and inherited from the mother who suffered from mild leuko-neutropenia. Both patients showed lower SRSF4 protein expression and altered mitochondrial function and energetic metabolism in primary lymphocytes and Epstein-Barr-virus (EBV)-immortalized lymphoblasts compared to healthy donor (HD) cells, which appeared associated with low mTOR phosphorylation and an imbalance in the proteins regulating mitochondrial biogenesis (i.e., CLUH) and dynamics (i.e., DRP1 and OPA1). Transfection with the wtSRSF4 gene restored mitochondrial function. In conclusion, this study shows that the described variant of the SRSF4 gene is pathogenetic and causes reduced SRSF4 protein expression, which leads to mitochondrial dysfunction. Since mitochondrial function is crucial for hematopoietic stem cell maintenance and some genetic bone marrow failure syndromes display mitochondrial defects, the SRSF4 mutation could have substantially contributed to the clinical phenotype of our patient.

Partial correction of immunodeficiency by lentiviral vector gene therapy in mouse models carrying Rag1 hypomorphic mutations.

Introduction: Recombination activating genes (RAG) 1 and 2 defects are the most frequent form of severe combined immunodeficiency (SCID). Patients with residual RAG activity have a spectrum of clinical manifestations ranging from Omenn syndrome to delayed-onset combined immunodeficiency, often associated with granulomas and/or autoimmunity (CID-G/AI). Lentiviral vector (LV) gene therapy (GT) has been proposed as an alternative treatment to the standard hematopoietic stem cell transplant and a clinical trial for RAG1 SCID patients recently started. However, GT in patients with hypomorphic RAG mutations poses additional risks, because of the residual endogenous RAG1 expression and the general state of immune dysregulation and associated inflammation. Methods: In this study, we assessed the efficacy of GT in 2 hypomorphic Rag1 murine models (Rag1F971L/F971L and Rag1R972Q/R972Q), exploiting the same LV used in the clinical trial encoding RAG1 under control of the MND promoter. Results and discussion: Starting 6 weeks after transplant, GT-treated mice showed a decrease in proportion of myeloid cells and a concomitant increase of B, T and total white blood cells. However, counts remained lower than in mice transplanted with WT Lin- cells. At euthanasia, we observed a general redistribution of immune subsets in tissues, with the appearance of mature recirculating B cells in the bone marrow. In the thymus, we demonstrated correction of the block at double negative stage, with a modest improvement in the cortical/medullary ratio. Analysis of antigenspecific IgM and IgG serum levels after in vivo challenge showed an amelioration of antibody responses, suggesting that the partial immune correction could confer a clinical benefit. Notably, no overt signs of autoimmunity were detected, with B-cell activating factor decreasing to normal levels and autoantibodies remaining stable after GT. On the other hand, thymic enlargement was frequently observed, although not due to vector integration and insertional mutagenesis. In conclusion, our work shows that GT could partially alleviate the combined immunodeficiency of hypomorphic RAG1 patients and that extensive efficacy and safety studies with alternative models are required before commencing RAG gene therapy in thesehighly complex patients.

Computational intelligence analysis of high-risk neuroblastoma patient health records reveals time to maximum response as one of the most relevant factors for outcome prediction.

OBJECTIVE: Seek new candidate prognostic markers for neuroblastoma outcome, relapse or progression. MATERIALS AND METHODS: In this multicentre and retrospective study, Random Forests coupled with recursive feature elimination techniques were applied to electronic records (55 clinical features) of 3034 neuroblastoma patients. To assess model performance and feature importance, dataset was split into a training set (80%) and a test set (20%). RESULTS: In the test set, the mean Matthews correlation coefficient for the Random Forests models was greater than 0.46. Feature importance analysis revealed that, together with maximum response to first-line treatment (D_MAX_RESP), time to maximum response to first-line treatment (TIME_MAX_RESP.days) is a relevant predictor of both patients' outcome and relapse\progression. We showed the prognostic value of the max response to first-line treatment in clinically relevant subsets of high-, intermediate-, and low-risk patients for both overall and relapse-free survival (Log-rank p-value<0.0001). In high-risk patients older than 18 months and stage 4 tumour achieving a complete response or very good partial response, patients who exhibited a D_MAX_RESP greater than 9 months showed a better prognosis with respect to patients achieving D_MAX_RESP earlier than 9 months (overall survival): hazard ratio 3.3 95% confidence interval 1.8-5.9, Log-rank p-value p < 0.0001; relapse-free survival: 3.2 95%CI 1.8-5.6, Log-rank p-value p < 0.0001). CONCLUSION: Our findings evidence the emerging role of the TIME_MAX_RESP.days in addition to the D_MAX_RESP as relevant predictors of outcome and relapse\progression in neuroblastoma with potential clinical impact on the management and treatment of patients.

Novel biallelic variants expand the phenotype of NAA20-related syndrome.

NAA20 is the catalytic subunit of the NatB complex, which is responsible for N-terminal acetylation of approximately 20% of the human proteome. Recently, pathogenic biallelic variants in NAA20 were associated with a novel neurodevelopmental disorder in five individuals with limited clinical information. We report two sisters harboring compound heterozygous variant (c.100C>T (p.Gln34Ter) and c.11T>C p.(Leu4Pro)) in the NAA20 gene, identified by exome sequencing. In vitro studies showed that the missense variant p.Leu4Pro resulted in a reduction of NAA20 catalytic activity due to weak coupling with the NatB auxiliary subunit. In addition, unpublished data of the previous families were reported, outlining the core phenotype of the NAA20-related disorder mostly characterized by cognitive impairment, microcephaly, ataxia, brain malformations, dysmorphism and variable occurrence of cardiac defect and epilepsy. Remarkably, our two patients featured epilepsy onset in adolescence suggesting this may be a part of syndrome evolution. Functional studies are needed to better understand the complexity of NAA20 variants pathogenesis as well as of other genes linked to N-terminal acetylation.

From FAIR4Health Project to 1+MG Initiative: A Spain - Italy Collaboration.

Results of two major projects funded by the European Union are taken into consideration: Fair4Health regarding the possibility of sharing clinical data in various environments applying FAIR principles and 1+Million Genome for the in-depth study of the human genome in Europe. Specifically, the Gaslini hospital plans to move on both areas joining the Hospital on FHIR initiative matured within the fair4health project and also collaborate with other Italian healthcare facilities through the implementation of a Proof of Concept (PoC) in the 1+MG. The aim of this short paper is to evaluate the applicability of some of the tools of the fair4health project to the Gaslini infrastructure to facilitate its participation in the PoC. One of the aims is also to prove the possibility of reuse the results of well-performed European funded projects to boost routine research in qualified healthcare facilities.

Moyamoya Vasculopathy in Neurofibromatosis Type 1 Pediatric Patients: The Role of Rare Variants of RNF213.

Neurofibromatosis type 1 (NF1) is a neurocutaneous disorder caused by mutations in NF1 gene, coding for neurofibromin 1. NF1 can be associated with Moyamoya disease (MMD), and this association, typical of paediatric patients, is referred to as Moyamoya syndrome (MMS). MMD is a cerebral arteriopathy characterized by the occlusion of intracranial arteries and collateral vessel formation, which increase the risk of ischemic and hemorrhagic events. RNF213 gene mutations have been associated with MMD, so we investigated whether rare variants of RNF213 could act as genetic modifiers of MMS phenotype in a pediatric cohort of 20 MMS children, 25 children affected by isolated MMD and 47 affected only by isolated NF1. By next-generation re-sequencing (NGS) of patients' DNA and gene burden tests, we found that RNF213 seems to play a role only for MMD occurrence, while it does not appear to be involved in the increased risk of Moyamoya for MMS patients. We postulated that the loss of neurofibromin 1 can be enough for the excessive proliferation of vascular smooth muscle cells, causing Moyamoya arteriopathy associated with NF1. Further studies will be crucial to support these findings and to elucidate the possible role of other genes, enhancing our knowledge about pathogenesis and treatment of MMS.

Deciphering clinical significance of BCL11A isoforms and protein expression roles in triple-negative breast cancer subtype.

PURPOSE: Triple negative breast cancer (TNBC) is an aggressive clinical tumor, accounting for about 25% of breast cancer (BC) related deaths. Chemotherapy is the only therapeutic option to treat TNBC, hence a detailed understanding of the biology and its categorization is required. To investigate the clinical relevance of BCL11A in TNBC subtype, we focused on gene and protein expression and its mutational status in a large cohort of this molecular subtype. METHODS: Gene expression profiling of BCL11A and its isoforms (BCL11A-XL, BCL11A-L and BCL11A-S) has been determined in Luminal A, Luminal B, HER2-enriched and TNBC subtypes. BCL11A protein expression has been analyzed by immunohistochemistry (IHC) and its mutational status by Sanger sequencing. RESULTS: In our study, BCL11A was significantly overexpressed in TNBC both at transcriptional and translational levels compared to other BC molecular subtypes. A total of 404 TNBCs were selected and examined showing a high prevalence of BCL11A-XL (37.3%) and BCL11A-L (31.4%) isoform expression in TNBC, associated with a 26% of BCL11A protein expression levels. BCL11A protein expression predicts scarce LIV (HR = 0.52; 95% CI, 0.29-0.92, P = 0.03) and AR downregulation (HR = 0.37; 95% CI, 0.16-0.88; P = 0.02), as well as a higher proliferative index in TNBC cells. BCL11A-L expression is associated with more aggressive TNBC histological types, such as medullary and metaplastic carcinoma. CONCLUSION: Our finding showed that BCL11A protein expression acts as an unfavorable prognostic factor in TNBC patients, especially in non luminal TNBCs subgroups. These results may yield a better treatment strategy by providing a new parameter for TNBC classification.

Exploration of Tools for the Interpretation of Human Non-Coding Variants.

The advent of Whole Genome Sequencing (WGS) broadened the genetic variation detection range, revealing the presence of variants even in non-coding regions of the genome, which would have been missed using targeted approaches. One of the most challenging issues in WGS analysis regards the interpretation of annotated variants. This review focuses on tools suitable for the functional annotation of variants falling into non-coding regions. It couples the description of non-coding genomic areas with the results and performance of existing tools for a functional interpretation of the effect of variants in these regions. Tools were tested in a controlled genomic scenario, representing the ground-truth and allowing us to determine software performance.

VariantAlert: A web-based tool to notify updates in genetic variant annotations.

The reinterpretation of variants based on updated annotations is part of the routine work of research laboratories: the more data is collected about a specific variant, the higher the probability to reinterpret its classification. To support this task, we developed VariantAlert, a web-based tool to help researchers and clinicians to be constantly informed about changes in variant annotations extracted from multiple sources. VariantAlert provides daily re-annotation of variants using external resources accessed through application programming interface, such as MyVariant.info providing in turn links to gnomAD, catalogue of somatic mutations In cancer (COSMIC), ClinVar, CIViC, and many others. Researchers and clinicians can submit one or more lists of variants. If a change is detected for the annotation of a variant due to the upgrade of the underlying resource (e.g., change in gnomAD allele frequency, presence in COSMIC database, change in ClinVar classification) the user is notified by email and updated annotations are stored on the web-site. VariantAlert is freely available at https://github.com/next-crs4/VariantAlert. Installation and deployment are easy thanks to the use of the Docker platform. A Makefile allows you to easily bootstrap VariantAlert. VariantAlert is also available as a web service at https://variant-alert.crs4.it/.

Spontaneous NLRP3 inflammasome-driven IL-1-ß secretion is induced in severe COVID-19 patients and responds to anakinra treatment.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection may result in a severe pneumonia associated with elevation of blood inflammatory parameters, reminiscent of cytokine storm syndrome. Steroidal anti-inflammatory therapies have shown efficacy in reducing mortality in critically ill patients; however, the mechanisms by which SARS-CoV-2 triggers such an extensive inflammation remain unexplained. OBJECTIVES: To dissect the mechanisms underlying SARS-CoV-2-associated inflammation in patients with severe coronavirus disease 2019 (COVID-19), we studied the role of IL-1ß, a pivotal cytokine driving inflammatory phenotypes, whose maturation and secretion are regulated by inflammasomes. METHODS: We analyzed nod-like receptor protein 3 pathway activation by means of confocal microscopy, plasma cytokine measurement, cytokine secretion following in vitro stimulation of blood circulating monocytes, and whole-blood RNA sequencing. The role of open reading frame 3a SARS-CoV-2 protein was assessed by confocal microscopy analysis following nucleofection of a monocytic cell line. RESULTS: We found that circulating monocytes from patients with COVID-19 display ASC (adaptor molecule apoptotic speck like protein-containing a CARD) specks that colocalize with nod-like receptor protein 3 inflammasome and spontaneously secrete IL-1ß in vitro. This spontaneous activation reverts following patient's treatment with the IL-1 receptor antagonist anakinra. Transfection of a monocytic cell line with cDNA coding for the ORF3a SARS-CoV-2 protein resulted in ASC speck formation. CONCLUSIONS: These results provide further evidence that IL-1ß targeting could represent an effective strategy in this disease and suggest a mechanistic explanation for the strong inflammatory manifestations associated with COVID-19.

Untargeted LC-HRMS Based-Plasma Metabolomics Reveals 3-O-Methyldopa as a New Biomarker of Poor Prognosis in High-Risk Neuroblastoma.

Neuroblastoma (NB) is the most common extracranial malignant tumor in children. Although the survival rate of NB has improved over the years, the outcome of NB still remains poor for over 30% of cases. A more accurate risk stratification remains a key point in the study of NB and the availability of novel prognostic biomarkers of "high-risk" at diagnosis could help improving patient stratification and predicting outcome. In this paper we show a biomarker discovery approach applied to the plasma of 172 NB patients. Plasma samples from a first cohort of NB patients and age-matched healthy controls were used for untargeted metabolomics analysis based on high-resolution mass spectrometry (HRMS). Differential expression analysis highlighted a number of metabolites annotated with a high degree of identification. Among them, 3-O-methyldopa (3-O-MD) was validated in a second cohort of NB patients using a targeted metabolite profiling approach and its prognostic potential was also analyzed by survival analysis on patients with 3 years follow-up. High expression of 3-O-MD was associated with worse prognosis in the subset of patients with stage M tumor (log-rank p < 0.05) and, among them, it was confirmed as a prognostic factor able to stratify high-risk patients older than 18 months. 3-O-MD might be thus considered as a novel prognostic biomarker of NB eligible to be included at diagnosis among catecholamine metabolite panels in prospective clinical studies. Further studies are warranted to exploit other potential biomarkers highlighted using our approach.

Gut Microbiota Markers and Dietary Habits Associated with Extreme Longevity in Healthy Sardinian Centenarians.

This study was aimed at characterizing the gut microbiota (GM) and its functional profile in two groups of Sardinian subjects with a long healthy life expectancy, overall named Long-Lived Subjects (LLS) [17 centenarians (CENT) and 29 nonagenarians (NON)] by comparing them to 46 healthy younger controls (CTLs). In addition, the contribution of genetics and environmental factors to the GM phenotype was assessed by comparing a subgroup of seven centenarian parents (CPAR) with a paired cohort of centenarians' offspring (COFF). The analysis was performed through Next Generation Sequencing (NGS) of the V3 and V4 hypervariable region of the 16S rRNA gene on the MiSeq Illumina platform. The Verrucomicrobia phylum was identified as the main biomarker in CENT, together with its members Verrucomicrobiaceae, Akkermansia and Akkermansia muciniphila. In NON, the strongest associations concern Actinobacteria phylum, Bifidobacteriaceae and Bifidobacterium, while in CTLs were related to the Bacteroidetes phylum, Bacteroidaceae, Bacteroides and Bacteroides spp. Intestinal microbiota of CPAR and COFF did not differ significantly from each other. Significant correlations between bacterial taxa and clinical and lifestyle data, especially with Mediterranean diet adherence, were observed. We observed a harmonically balanced intestinal community structure in which the increase in taxa associated with intestinal health would limit and counteract the action of potentially pathogenic bacterial species in centenarians. The GM of long-lived individuals showed an intrinsic ability to adapt to changing environmental conditions, as confirmed by functional analysis. The GM analysis of centenarians' offspring suggest that genetics and environmental factors act synergistically as a multifactorial cause in the modulation of GM towards a phenotype similar to that of centenarians, although these findings need to be confirmed by larger study cohorts and by prospective studies.

De novo truncating NOVA2 variants affect alternative splicing and lead to heterogeneous neurodevelopmental phenotypes.

Alternative splicing (AS) is crucial for cell-type-specific gene transcription and plays a critical role in neuronal differentiation and synaptic plasticity. De novo frameshift variants in NOVA2, encoding a neuron-specific key splicing factor, have been recently associated with a new neurodevelopmental disorder (NDD) with hypotonia, neurological features, and brain abnormalities. We investigated eight unrelated individuals by exome sequencing (ES) and identified seven novel pathogenic NOVA2 variants, including two with a novel localization at the KH1 and KH3 domains. In addition to a severe NDD phenotype, novel clinical features included psychomotor regression, attention deficit-hyperactivity disorder (ADHD), dyspraxia, and urogenital and endocrinological manifestations. To test the effect of the variants on splicing regulation, we transfected HeLa cells with wildtype and mutant NOVA2 complementary DNA (cDNA). The novel variants NM_002516.4:c.754_756delCTGinsTT p.(Leu252Phefs*144) and c.1329dup p.(Lys444Glnfs*82) all negatively affected AS events. The distal p.(Lys444Glnfs*82) variant, causing a partial removal of the KH3 domain, had a milder functional effect leading to an intermediate phenotype. Our findings expand the molecular and phenotypic spectrum of NOVA2-related NDD, supporting the pathogenic role of AS disruption by truncating variants and suggesting that this is a heterogeneous condition with variable clinical course.

A Phenotypic-Driven Approach for the Diagnosis of WOREE Syndrome.

Background: WOREE syndrome is a rare neurodevelopmental disorder featuring drug-resistant epilepsy and global developmental delay. The disease, caused by biallelic pathogenic variants in the WWOX gene, usually leads to severe disability or death within the first years of life. Clinicians have become more confident with the phenotypic picture of WOREE syndrome, allowing earlier clinical diagnosis. We report a boy with a peculiar clinic-radiological pattern supporting the diagnosis of WOREE syndrome. Methods: DNA was extracted from blood samples of the proband and his parents and subjected to Exome Sequencing (ES). Agarose gel electrophoresis, real-time quantitative PCR (Q-PCR), and array-CGH 180K were also performed. Results: ES detected a pathogenic stop variant (c.790C > T, p.Arg264*) in one allele of WWOX in the proband and his unaffected mother. A 180K array-CGH analysis revealed a 84,828-bp (g.chr16:78,360,803-78,445,630) deletion encompassing exon 6. The Q-PCR product showed that the proband and his father harbored the same deleted fragment, fusing exons 5 and 7 of WWOX. Conclusions: Genetic testing remains crucial in establishing the definitive diagnosis of WOREE syndrome and allows prenatal interventions/parental counseling. However, our findings suggest that targeted Next Generation Sequencing-based testing may occasionally show technical pitfalls, prompting further genetic investigation in selected cases with high clinical suspicion.

Circulating microRNAs as novel non-invasive biomarkers of paediatric celiac disease and adherence to gluten-free diet.

BACKGROUND: Celiac Disease (CD) is a multifactorial autoimmune enteropathy (with a prevalence of approximately 1% worldwide) that exhibits a wide spectrum of clinical, serological and histological manifestations. For the diagnosis of paediatric CD, the gold standard is the combination of serological tests (with high TGA-IgA values greater than 10 times the upper limit of normal) and duodenal biopsy (with a positive TGA-IgA but low titer). Therefore, a diagnostic test that totally excludes an invasive approach has not been discovered so far and the discovery of novel biological markers would represent an undoubted advantage for the diagnosis of CD and prognostic evaluation. MicroRNAs (miRNAs), small non-coding RNAs (18-22 nucleotides) that regulate gene expression at post-transcriptional level and play important roles in many biological processes, represent a novel class of potential disease biomarkers. Their presence in biological fluids (i.e., serum, plasma, saliva, urine) provides the opportunity to employ circulating miRNAs as novel non-invasive biomarkers. METHODS: In our prospective observational study, we examined the expression of circulating miRNAs in a cohort of CD patients (both at diagnosis and on gluten-free diet, respectively referred as CD and GFD) compared to healthy controls. By small RNA-Seq we discovered a set of circulating miRNAs that were further validated by qPCR with specific assays. FINDINGS: We found that out of the 13 miRNAs able to discriminate the three groups (i.e., CD, GFD and controls), three of them, namely miR-192-5p, miR-215-5p and miR-125b-5p (alone or in combination), were able to discriminate these three groups with high accuracy and specificity. INTERPRETATION: Our conclusions emphasize that these circulating miRNAs can be employed not only for the diagnosis of CD patients with a low TGA-IgA titer but also to monitor the adherence to a gluten-free diet by CD patients. In conclusion, we suggest the use of the circulating miRNAs identified in this work as a novel diagnostic and follow-up tool for paediatric CD. FUNDING: This work was supported by Fondazione Celiachia Onlus (FC) Grant n° 018/FC/2013 and by Italian Ministry of Health (Ricerca Corrente).

Laniakea@ReCaS: exploring the potential of customisable Galaxy on-demand instances as a cloud-based service.

BACKGROUND: Improving the availability and usability of data and analytical tools is a critical precondition for further advancing modern biological and biomedical research. For instance, one of the many ramifications of the COVID-19 global pandemic has been to make even more evident the importance of having bioinformatics tools and data readily actionable by researchers through convenient access points and supported by adequate IT infrastructures. One of the most successful efforts in improving the availability and usability of bioinformatics tools and data is represented by the Galaxy workflow manager and its thriving community. In 2020 we introduced Laniakea, a software platform conceived to streamline the configuration and deployment of "on-demand" Galaxy instances over the cloud. By facilitating the set-up and configuration of Galaxy web servers, Laniakea provides researchers with a powerful and highly customisable platform for executing complex bioinformatics analyses. The system can be accessed through a dedicated and user-friendly web interface that allows the Galaxy web server's initial configuration and deployment. RESULTS: "Laniakea@ReCaS", the first instance of a Laniakea-based service, is managed by ELIXIR-IT and was officially launched in February 2020, after about one year of development and testing that involved several users. Researchers can request access to Laniakea@ReCaS through an open-ended call for use-cases. Ten project proposals have been accepted since then, totalling 18 Galaxy on-demand virtual servers that employ ~ 100 CPUs, ~ 250 GB of RAM and ~ 5 TB of storage and serve several different communities and purposes. Herein, we present eight use cases demonstrating the versatility of the platform. CONCLUSIONS: During this first year of activity, the Laniakea-based service emerged as a flexible platform that facilitated the rapid development of bioinformatics tools, the efficient delivery of training activities, and the provision of public bioinformatics services in different settings, including food safety and clinical research. Laniakea@ReCaS provides a proof of concept of how enabling access to appropriate, reliable IT resources and ready-to-use bioinformatics tools can considerably streamline researchers' work.