Explore the vast range of titles available.

Alport syndrome (AS) is an inherited genetic disorder characterized by range of alterations from glomerular basement membrane abnormalities up to end-stage renal disease. Pathogenic variants in the collagen α3, α4, and α5 encoding genes are causative both of the autosomal dominant and of the X-linked forms of AS. Podocytes are the only renal cells that are able to produce the COL(IV)a3-a4a5 heterotrimer. We have previously demonstrated how it is possible to isolate podocyte-lineage cells from urine of patients, providing an easily accessible cellular model closer to the podocytes’ physiological conditions. Taking advantage of disease-relevant cell lines, we employed a two-plasmid approach in order to achieve a beneficial and stable variant-specific correction using CRISPR/Cas9 genome editing. One plasmid carries a Donor DNA and a reporter system mCherry/GFP to track the activity of Cas9 in cells. The other plasmid carries a self-cleaving SpCas9 and the variant-specific sgRNA. We have analyzed two stable podocyte-lineage cell lines, harboring a variant in the X-linked COL4A5 (p.(Gly624Asp)) and in the autosomal COL4A3 gene (p.(Gly856Glu)). We have achieved reversion of variants greater than 40% with undesired insertions/deletions lower than 15%. Overall, we have demonstrated a new gene therapy approach directly on patients’ cells, key players of Alport pathogenesis, and we have reverted COL4 causative variants towards the wild type state. These results, in combination with preclinical models, could open new frontiers in the management and the treatment of the disorder.

Variations in the Forkhead Box G1 (FOXG1) gene cause FOXG1 syndrome spectrum, including the congenital variant of Rett syndrome, characterized by early onset of regression, Rett-like and jerky movements, and cortical visual impairment. Due to the largely unknown pathophysiological mechanisms downstream the impairment of this transcriptional regulator, a specific treatment is not yet available. Since both haploinsufficiency and hyper-expression of FOXG1 cause diseases in humans, we reasoned that adding a gene under nonnative regulatory sequences would be a risky strategy as opposed to a genome editing approach where the mutated gene is reversed into wild-type. Here, we demonstrate that an adeno-associated viruses (AAVs)-coupled CRISPR/Cas9 system is able to target and correct FOXG1 variants in patient-derived fibroblasts, induced Pluripotent Stem Cells (iPSCs) and iPSC-derived neurons. Variant-specific single-guide RNAs (sgRNAs) and donor DNAs have been selected and cloned together with a mCherry/EGFP reporter system. Specific sgRNA recognition sequences were inserted upstream and downstream Cas9 CDS to allow self-cleavage and inactivation. We demonstrated that AAV serotypes vary in transduction efficiency depending on the target cell type, the best being AAV9 in fibroblasts and iPSC-derived neurons, and AAV2 in iPSCs. Next-generation sequencing (NGS) of mCherry+/EGFP+ transfected cells demonstrated that the mutated alleles were repaired with high efficiency (20–35% reversion) and precision both in terms of allelic discrimination and off-target activity. The genome editing strategy tested in this study has proven to precisely repair FOXG1 and delivery through an AAV9-based system represents a step forward toward the development of a therapy for Rett syndrome.

Rett syndrome is a progressive neurodevelopmental disorder which affects almost exclusively girls, caused by variants in MECP2 gene. Effective therapies for this devastating disorder are not yet available and the need for tight regulation of MECP2 expression for brain to properly function makes gene replacement therapy risky. For this reason, gene editing with CRISPR/Cas9 technology appears as a preferable option for the development of new therapies. To study the disease, we developed and characterized a human neuronal model obtained by genetic reprogramming of patient-derived primary fibroblasts into induced Pluripotent Stem Cells. This cellular model represents an important source for our studies, aiming to correct MECP2 variants in neurons which represent the primarily affected cell type. We engineered a gene editing toolkit composed by a two-plasmid system to correct a hotspot missense variant in MECP2, c.473 C > T (p.(Thr158Met)). The first construct expresses the variant-specific sgRNA and the Donor DNA along with a fluorescent reporter system. The second construct brings Cas9 and targets for auto-cleaving, to avoid long-term Cas9 expression. NGS analysis on sorted cells from four independent patients demonstrated an exceptionally high editing efficiency, with up to 80% of HDR and less than 1% of indels in all patients, outlining the relevant potentiality of the approach for Rett syndrome therapy.

Efficient humoral responses rely on DNA damage, mutagenesis and error-prone DNA repair. Diversification of B cell receptors through somatic hypermutation and class-switch recombination are initiated by cytidine deamination in DNA mediated by activation-induced cytidine deaminase (AID) 1 and by the subsequent excision of the resulting uracils by uracil DNA glycosylase (UNG) and by mismatch repair proteins 1 – 3 . Although uracils arising in DNA are accurately repaired 1 – 4 , how these pathways are co-opted to generate mutations and double-strand DNA breaks in the context of somatic hypermutation and class-switch recombination is unknown 1 – 3 . Here we performed a genome-wide CRISPR–Cas9 knockout screen for genes involved in class-switch recombination and identified FAM72A, a protein that interacts with the nuclear isoform of UNG (UNG2) 5 and is overexpressed in several cancers 5 . We show that the FAM72A–UNG2 interaction controls the levels of UNG2 and that class-switch recombination is defective in Fam72a −/− B cells due to the upregulation of UNG2. Moreover, we show that somatic hypermutation is reduced in Fam72a −/− B cells and that its pattern is skewed upon upregulation of UNG2. Our results are consistent with a model in which FAM72A interacts with UNG2 to control its physiological level by triggering its degradation, regulating the level of uracil excision and thus the balance between error-prone and error-free DNA repair. Our findings have potential implications for tumorigenesis, as reduced levels of UNG2 mediated by overexpression of Fam72a would shift the balance towards mutagenic DNA repair, rendering cells more prone to acquire mutations. FAM72A interacts with UNG2 to regulate the balance between error-prone and error-free DNA repair.

The combined impact of common and rare exonic variants in COVID-19 host genetics is currently insufficiently understood. Here, common and rare variants from whole-exome sequencing data of about 4000 SARS-CoV-2-positive individuals were used to define an interpretable machine-learning model for predicting COVID-19 severity. First, variants were converted into separate sets of Boolean features, depending on the absence or the presence of variants in each gene. An ensemble of LASSO logistic regression models was used to identify the most informative Boolean features with respect to the genetic bases of severity. The Boolean features selected by these logistic models were combined into an Integrated PolyGenic Score that offers a synthetic and interpretable index for describing the contribution of host genetics in COVID-19 severity, as demonstrated through testing in several independent cohorts. Selected features belong to ultra-rare, rare, low-frequency, and common variants, including those in linkage disequilibrium with known GWAS loci. Noteworthily, around one quarter of the selected genes are sex-specific. Pathway analysis of the selected genes associated with COVID-19 severity reflected the multi-organ nature of the disease. The proposed model might provide useful information for developing diagnostics and therapeutics, while also being able to guide bedside disease management.

Background Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is a novel coronavirus that caused an ongoing pandemic of a pathology termed Coronavirus Disease 19 (COVID-19). Several studies reported that both COVID-19 and RTEL1 variants are associated with shorter telomere length, but a direct association between the two is not generally acknowledged. Here we demonstrate that up to 8.6% of severe COVID-19 patients bear RTEL1 ultra-rare variants, and show how this subgroup can be recognized. Methods A cohort of 2246 SARS-CoV-2-positive subjects, collected within the GEN-COVID Multicenter study, was used in this work. Whole exome sequencing analysis was performed using the NovaSeq6000 System, and machine learning methods were used for candidate gene selection of severity. A nested study, comparing severely affected patients bearing or not variants in the selected gene, was used for the characterisation of specific clinical features connected to variants in both acute and post-acute phases. Results Our GEN-COVID cohort revealed a total of 151 patients carrying at least one RTEL1 ultra-rare variant, which was selected as a specific acute severity feature. From a clinical point of view, these patients showed higher liver function indices, as well as increased CRP and inflammatory markers, such as IL-6. Moreover, compared to control subjects, they present autoimmune disorders more frequently. Finally, their decreased diffusion lung capacity for carbon monoxide after six months of COVID-19 suggests that RTEL1 variants can contribute to the development of SARS-CoV-2-elicited lung fibrosis. Conclusion RTEL1 ultra-rare variants can be considered as a predictive marker of COVID-19 severity, as well as a marker of pathological evolution in pulmonary fibrosis in the post-COVID phase. This notion can be used for a rapid screening in hospitalized infected people, for vaccine prioritization, and appropriate follow-up assessment for subjects at risk. Trial Registration NCT04549831 ( www.clinicaltrial.org )

We employed a multifaceted computational strategy to identify the genetic factors contributing to increased risk of severe COVID-19 infection from a Whole Exome Sequencing (WES) dataset of a cohort of 2000 Italian patients. We coupled a stratified k -fold screening, to rank variants more associated with severity, with the training of multiple supervised classifiers, to predict severity based on screened features. Feature importance analysis from tree-based models allowed us to identify 16 variants with the highest support which, together with age and gender covariates, were found to be most predictive of COVID-19 severity. When tested on a follow-up cohort, our ensemble of models predicted severity with high accuracy (ACC = 81.88%; AUCROC = 96%; MCC = 61.55%). Our model recapitulated a vast literature of emerging molecular mechanisms and genetic factors linked to COVID-19 response and extends previous landmark Genome-Wide Association Studies (GWAS). It revealed a network of interplaying genetic signatures converging on established immune system and inflammatory processes linked to viral infection response. It also identified additional processes cross-talking with immune pathways, such as GPCR signaling, which might offer additional opportunities for therapeutic intervention and patient stratification. Publicly available PheWAS datasets revealed that several variants were significantly associated with phenotypic traits such as “Respiratory or thoracic disease”, supporting their link with COVID-19 severity outcome. A multifaceted computational strategy identifies 16 genetic variants contributing to increased risk of severe COVID-19 infection from a Whole Exome Sequencing dataset of a cohort of Italian patients.

Background: This study aimed to review the data available in the current literature concerning the complications and recurrence of instability following medial patellofemoral ligament (MPFL) reconstruction for patellar instability in young and adolescent patients (those <20 years old). Methods: A systematic review was performed based on the preferred reporting items for systematic reviews and meta-analyses (PRISMA) guidelines. Two independent reviewers searched the PubMed, Scopus, EMBASE, and Cochrane databases. The terms “medial patellofemoral ligament” or “MPFL” and “reconstruction” and “young” or “adolescents” or “children” were used. The inclusion criteria for the literature review comprised studies that reported the complications and recurrences of instability in patients who had undergone MPFL reconstruction for patellar instability. Results: In all, 332 patients were included in the review, of which 195 were females (63.5%) and 112 were males (36.5%), and they totaled 352 treated knees. The mean age at the time of the surgery was 14.28 years, and the mean follow-up duration was 30.17 months. A total of 16 (4.5%) complications were reported: one (0.3%) patella fracture, one (0.3%) screw removal due to intolerance, one (0.3%) infection, five (1.4%) wound complications, six (1.7%) subluxations and two (0.6%) instances of post-operative stiffness. A total of 18 (5.1%) recurrences of patellar instability were recorded. Conclusions: MPFL reconstruction in young patients can be considered an effective and safe treatment leading to clinical improvement in terms of recurrence of dislocation. No major complications related to the technique were reported, but a high level of research evidence is required to better evaluate the clinical results in a long-term follow-up.