Background: The expression of imprinted genes, which depends on their gamete of origin, is regulated by DNA sequences characterized by differential methylation between the maternal and paternal alleles (also known as germline differentially methylated regions or gDMRs). The most common molecular defect associated with Beckwith-Wiedemann syndrome (BWS), a condition linked to overgrowth and tumours, is the loss of methylation of the KCNQ1OT1-TSS gDMR located on chromosome 11p15.5 (also known as IC2 LoM). Approximately one-third of BWS patients with IC2 LoM exhibit multi-locus imprinting disturbances (MLID). While maternal-effect variants in proteins of the oocyte subcortical maternal complex (SCMC) have been linked to MLID, the underlying mechanisms and health impact of this epigenetic disturbance remain unclear. Results: We used the Infinium EPIC methylation array to investigate whole-genome CpG methylation in 64 BWS patients with IC2 LoM and 37 control subjects. We distinguished two patient groups, one with a variable methylation level of 24 gDMRs and the other with single-locus IC2 LoM. We observed that the mothers of the former patient group carried more variants in maternal-effect genes than those of the latter group, and 50% of them, but none of the latter group had variants in the SCMC genes. Additionally, in the former group, the mothers were older at the time of pregnancy, and the patients showed higher variation in methylation levels of thousands of CpGs located in non-imprinted loci, including protochaderins and cancer-associated genes. We found no differences in clinical features or in the incidence of assisted reproductive technology between the two patient groups. However, multiple affected siblings and recurrent miscarriages were observed only among cases with biallelic maternal-effect SCMC gene variants. Conclusions: This study demonstrates that the BWS patients with MLID exhibit highly variable methylation changes that affect both imprinted and non-imprinted loci in a seemingly stochastic manner throughout the genome. These findings support the hypothesis that MLID results from the interaction of maternal-effect genes and environmental factors in aged oocytes, leading to disordered DNA methylation in the whole genome. Future research should investigate whether and how these epimutations impact the health of affected individuals, particularly in adulthood.

The immune regulatory defects that promote neuroinflammation in multiple sclerosis (MS) remain unclear. We show that a specific regulatory T (Treg) cell subpopulation expressing Notch3 was increased in individuals with MS and in mice with experimental autoimmune encephalomyelitis (EAE). Notch3+ Treg cells were induced by the gut microbiota via Toll-like receptor (TLR)-dependent mechanisms. They then translocated to the central nervous system (CNS) in EAE where they promoted disease severity. Notch3 interacted with delta-like ligand 1 (DLL1) on microglia to subvert Treg cells into T helper 17 (Th17) cells. Notch3 deletion in Treg cells prevented EAE onset by stabilizing Treg cells and by simultaneously promoting the expansion of a tissue-resident Treg cell population that expressed neuropeptide Y receptor 1 (NPY1R) and which suppressed pathogenic IFN-γ+ and GM-CSF+ T cells. Our studies thus identify altered Treg cell population dynamics as a fundamental pathogenic mechanism in autoimmune neuroinflammation.

Background: Genomic imprinting is required for normal development, and abnormal methylation of differentially methylated regions (iDMRs) controlling the parent of origin-dependent expression of the imprinted genes has been found in congenital disorders affecting growth, metabolism, neurobehavior, and in cancer. In most of these cases the cause of the imprinting abnormalities is unknown. Also, these studies have generally been performed on a limited number of CpGs, and a systematic investigation of iDMR methylation in the general population is lacking. Results: By analysing a vast number of either in-house generated or online available whole-genome methylation array datasets of unaffected individuals, and patients with complex and rare disorders, we determined the most common iDMR methylation profiles in a large population and identified many genetic and non-genetic factors contributing to their variability in blood DNA. We found that methylation variability was not homogeneous within the iDMRs and that the CpGs closer to the ZFP57 binding sites are less susceptible to methylation changes. We demonstrated the methylation polymorphism of three iDMRs and the atypical behaviour of several others, and reported the association of 25 disease- and 47 non-disease-complex traits as well as 15 Mendelian and chromosomal disorders with iDMR methylation changes. The most significantly associated complex traits included ageing, intracytoplasmic sperm injection, African versus European ancestry, female sex, pre- and postnatal exposure to pollutants and blood cell type compositions, while the associated genetic diseases included Down syndrome and the developmental disorders with molecular defects in the DNA methyltransferases DNMT1 and DNMT3B, H3K36 methyltransferase SETD2, chromatin remodelers SRCAP and SMARCA4 and transcription factor ADNP. Conclusions: These findings identify several genetic and non-genetic factors including new genes associated with genomic imprinting maintenance in humans, which may have a role in the aetiology of the diseases with imprinting abnormalities and have clear implications in molecular diagnostics.

Background: Splenomegaly represents a frequent non-infectious manifestation in Common Variable Immunodeficiency (CVID) and associates with specific clinical and immunophenotypic characteristics. Objective: To investigate the association between splenomegaly, infections, and immunophenotype in CVID patients. Methods: A cohort of 32 CVID patients (13 with splenomegaly) was enrolled. Infectious workup encompassed a detailed medical history and data derived from routine diagnostic assessments including specific virological analysis of blood and stool samples, and QuantiFERON assay for tuberculosis. Immunophenotype was assessed by multiparametric flow cytometry. Statistical analyses were performed using Prism and Jamovi software. Results: CMV viraemia was detected in 40 % of splenomegalic CVID (sCVID) and was absent in non-sCVID patients. Of all infectious agents, CMV was the only one associated with splenomegaly (p = 0.009). The inclusion of CMV replication as a causative factor for splenomegaly in CVID is in line with the knowledge that splenomegaly is a hallmark of acute CMV infection and could help explain in the present CVID cohort 75 % of otherwise unexplained splenomegalies. Flow cytometric analysis in sCVID vs. non-sCVID confirmed decreases in NK cell numbers and activation, in circulating inflammatory precursors (Lin−CD16+), and increased T cell activation as defined by HLA-DR/CD69/CD38 expression. Conclusion: Splenomegaly in CVID patients may associate also with CMV replication. The combined identification in CMV+ sCVID of NK cell, inflammatory precursor and T cell imbalances suggests a possible combined cellular defect at precursor level in a subset of sCVID patients. When integrated into everyday clinical management, CMV viraemia could become a useful additional parameter for patient characterization and stratification.

TBX1, a T-box transcription factor, is essential for pharyngeal apparatus development and marks cardiopharyngeal mesoderm (CPM) in various species. However, in mammals, we have an incomplete knowledge of the molecular pathways driving CPM diversification and of the role of TBX1 in this context. Using CPM-relevant in vitro differentiation of wild-type and Tbx1−/− mouse embryonic stem cells, we performed simultaneous single-nucleus RNA-seq and ATAC-seq at two stages, validated findings in embryos, and found that TBX1 loss affects gene expression and chromatin remodeling in a cell subpopulationspecific manner. TBX1 regulates chromatin accessibility and gene expression of distinct and evolutionarily conserved transcriptional modules for branchiomeric and cardiac development, and for tissue morphogenesis. Computational analyses predicted a feed-forward regulatory relationship between TBX1 and SIX factors. Notably, selected Tbx1 mutant CPM cell populations showed an altered differentiation trajectory, exhibiting activation of a mesothelial-like transcriptional program. We also observed cell death later in development. Thus, TBX1 is crucial for maintaining CPM transcriptional identity.

Electrical stimulation (ES) is widely employed in both clinical therapies and research settings where it has shown promise in promoting tissue regeneration, wound healing, and inflammation control. Research has also highlighted ES as a regulator of DNA demethylation, which plays a critical role in nerve regeneration and cellular repair mechanisms. While the impact of ES on epigenetic processes is recognized, its broader effects on cellular functions, particularly in inflammation and wound healing, are less understood. We recently showed how ES impacts inflammatory states by modulating transcriptomic and metabolomic profiles in a 3Din vitromodel where human fibroblasts and keratinocytes are included in a collagen matrix, i.e., even in the absence of the nervous system. Here, we propose to deepen our exploration on the differential effects on DNA methylation, including an investigation of the correlation with age acceleration using a mitotic clock. These results confirm and caution on the differential effect of DC on inflamed and non-inflamed samples and suggest an involvement of direct current stimuli at 1 V ((Formula presented.)) in the control of senescent processes associated with mitosis and inflammation; the mechanistic details of these will have to be clarified with additional experiments.

When collecting several data sets and heterogeneous data types on a given phenomenon of interest, the individual analysis of each data set will provide only a particular view of such phenomenon. Instead, integrating all the data may widen and deepen the results, offering a better view of the entire system. In the context of network integration, we propose the INet algorithm. INet assumes a similar network structure, representing latent variables in different network layers of the same system. Therefore, by combining individual edge weights and topological network structures, INet first constructs a Consensus Network that represents the shared information underneath the different layers to provide a global view of the entities that play a fundamental role in the phenomenon of interest. Then, it derives a Case Specific Network for each layer containing peculiar information of the single data type not present in all the others. We demonstrated good performance with our method through simulated data and detected new insights by analyzing biological and sociological datasets.

We propose AFTNet, a novel network-constraint survival analysis method based on the Weibull accelerated failure time (AFT) model solved by a penalized likelihood approach for variable selection and estimation. When using the log-linear representation, the inference problem becomes a structured sparse regression problem for which we explicitly incorporate the correlation patterns among predictors using a double penalty that promotes both sparsity and grouping effect. Moreover, we establish the theoretical consistency for the AFTNet estimator and present an efficient iterative computational algorithm based on the proximal gradient descent method. Finally, we evaluate AFTNet performance both on synthetic and real data examples.

Background: Osteoarthritis (OA) is a multifactorial, hypertrophic, and degenerative condition involving the whole joint and affecting a high percentage of middle-aged people. It is due to a combination of factors, although the pivotal mechanisms underlying the disease are still obscure. Moreover, current treatments are still poorly effective, and patients experience a painful and degenerative disease course. Methods: We used an integrative approach that led us to extract a consensus signature from a meta-analysis of three different OA cohorts. We performed a network-based drug prioritization to detect the most relevant drugs targeting these genes and validated in vitro the most promising candidates. We also proposed a risk score based on a minimal set of genes to predict the OA clinical stage from RNA-Seq data. Results: We derived a consensus signature of 44 genes that we validated on an independent dataset. Using network analysis, we identified Resveratrol, Tenoxicam, Benzbromarone, Pirinixic Acid, and Mesalazine as putative drugs of interest for therapeutics in OA for anti-inflammatory properties. We also derived a list of seven gene-targets validated with functional RT-qPCR assays, confirming the in silico predictions. Finally, we identified a predictive subset of genes composed of DNER, TNFSF11, THBS3, LOXL3, TSPAN2, DYSF, ASPN and HTRA1 to compute the patient's risk score. We validated this risk score on an independent dataset with a high AUC (0.875) and compared it with the same approach computed using the entire consensus signature (AUC 0.922). Conclusions: The consensus signature highlights crucial mechanisms for disease progression. Moreover, these genes were associated with several candidate drugs that could represent potential innovative therapeutics. Furthermore, the patient's risk scores can be used in clinical settings.

Motivation: methyLImp, a method we recently introduced for the missing value estimation of DNA methylation data, has demonstrated competitive performance in data imputation compared to the existing, general-purpose, approaches. However, imputation running time was considerably long and unfeasible in case of large datasets with numerous missing values. Results: methyLImp2 made possible computations that were previously unfeasible. We achieved this by introducing two important modifications that have significantly reduced the original running time without sacrificing prediction performance. First, we implemented a chromosome-wise parallel version of methyLImp. This parallelization reduced the runtime by several 10-fold in our experiments. Then, to handle large datasets, we also introduced a mini-batch approach that uses only a subset of the samples for the imputation. Thus, it further reduces the running time from days to hours or even minutes in large datasets.

Purpose: Common Variable Immunodeficiency (CVID) is characterized by hypogammaglobulinemia and failure of specific antibody production due to B-cell defects. However, studies have documented various T-cell abnormalities, potentially linked to viral complications. The frequency of Cytomegalovirus (CMV) replication in CVID cohorts is poorly studied. To address this gap in knowledge, we set up an observational study with the objectives of identifying CVID patients with active viraemia (CMV, Epstein-Barr virus (EBV)), evaluating potential correlations with immunophenotypic characteristics, clinical outcome, and the dynamic progression of clinical phenotypes over time. Methods: 31 CVID patients were retrospectively analysed according to viraemia, clinical and immunologic characteristics. 21 patients with non CVID humoral immunodeficiency were also evaluated as control. Results: Active viral replication of CMV and/or EBV was observed in 25% of all patients. CMV replication was detected only in CVID patients (16%). CVID patients with active viral replication showed reduced HLA-DR+ NK counts when compared with CMV-DNA negative CVID patients. Viraemic patients had lower counts of LIN−DNAMbright and LIN−CD16+ inflammatory lymphoid precursors which correlated with NK-cell subsets. Analysis of the dynamic progression of CVID clinical phenotypes over time, showed that the initial infectious phenotype progressed to complicated phenotypes with time. All CMV viraemic patients had complicated disease. Conclusion: Taken together, an impaired production of inflammatory precursors and NK activation is present in CVID patients with active viraemia. Since “Complicated” CVID occurs as a function of disease duration, there is need for an accurate evaluation of this aspect to improve classification and clinical management of CVID patients.

Background: Immune dysregulation and SARS-CoV-2 plasma viremia have been implicated in fatal COVID-19 disease. However, how these two factors interact to shape disease outcomes is unclear. Methods: We carried out viral and immunological phenotyping on a prospective cohort of 280 patients with COVID-19 presenting to acute care hospitals in Boston, Massachusetts and Genoa, Italy between June 1, 2020 and February 8, 2022. Disease severity, mortality, plasma viremia, and immune dysregulation were assessed. A mouse model of lethal H1N1 influenza infection was used to analyze the therapeutic potential of Notch4 and pyroptosis inhibition in disease outcome. Results: Stratifying patients based on %Notch4+ Treg cells and/or the presence of plasma viremia identified four subgroups with different clinical trajectories and immune phenotypes. Patients with both high %Notch4+ Treg cells and viremia suffered the most disease severity and 90-day mortality compared to the other groups even after adjusting for baseline comorbidities. Increased Notch4 and plasma viremia impacted different arms of the immune response in SARS-CoV-2 infection. Increased Notch4 was associated with decreased Treg cell amphiregulin expression and suppressive function whereas plasma viremia was associated with increased monocyte cell pyroptosis. Combinatorial therapies using Notch4 blockade and pyroptosis inhibition induced stepwise protection against mortality in a mouse model of lethal H1N1 influenza infection. Conclusions: The clinical trajectory and survival outcome in hospitalized patients with COVID-19 is predicated on two cardinal factors in disease pathogenesis: viremia and Notch4+ Treg cells. Intervention strategies aimed at resetting the immune dysregulation in COVID-19 by antagonizing Notch4 and pyroptosis may be effective in severe cases of viral lung infection.

Background: Aggressive malignancies, such as pancreatic cancer, are increasingly impacting young, female populations. Our investigation centered on whether the observed trends in cancer incidence were unique to pancreatic cancer or indicative of a broader trend across various cancer types. To delve deeper into this phenomenon, we analyzed cancer incidence trends across different age and sex groups. Furthermore, we explored differences in cancer incidence within specific young subgroups aged 18 to 26 and 27 to 34, to better understand the emerging incidence trend among young individuals. Methods: This study collected cancer incidence data from one of the Surveillance, Epidemiology, and End Results cancer registry databases (SEER22), with 10,183,928 total cases from 2000 to 2020. Data were analyzed through Joinpoint trend analysis approach to evaluate sex- and age-specific trends in cancer incidence. Exposure rates were reported as Average Annual Percentage Changes (AAPCs). Results: The analysis revealed significant age and sex-specific disparities, particularly among individuals aged 18–26 and 27–34. Pancreatic cancer incidence rates increased more in females aged 18–26 (AAPC, 9.37% [95% CI, 7.36–11.41%]; p <.0001) than in males (4.43% [95% CI, 2.36–6.53%]; p <.0001). Notably, among gender, age, and other malignancies, young females had the highest AAPCs for pancreatic cancer. Additionally, the incidence of gastric cancer, myeloma, and colorectal malignancies also showed higher AAPCs in young females compared to males. Conclusions: Recognizing emerging risk populations for highly lethal malignancies is crucial for early detection and effective disease management.

: The T-BOX transcription factor TBX1 is essential for the development of the pharyngeal apparatus and it is haploinsufficient in DiGeorge syndrome (DGS), a developmental anomaly associated with congenital heart disease and other abnormalities. The murine model recapitulates the heart phenotype and showed collagen accumulation. We first used a cellular model to study gene expression during cardiogenic differentiation of WT and Tbx1-/- mouse embryonic stem cells. Then we used a mouse model of DGS to test whether interfering with collagen accumulation using an inhibitor of lysyl hydroxylase would modify the cardiac phenotype of the mutant. We found that loss of Tbx1 in a precardiac differentiation model was associated with up regulation of a subset of ECM-related genes, including several collagen genes. In the in vivo model, early prenatal treatment with Minoxidil, a lysyl hydroxylase inhibitor, ameliorated the cardiac outflow tract septation phenotype in Tbx1 mutant fetuses, but it had no effect on septation in WT fetuses. We conclude that TBX1 suppresses a defined subset of ECM-related genes. This function is critical for OFT septation because the inhibition of collagen cross-linking in the mutant reduces significantly the penetrance of septation defects.

Endothelial cells (EC) differentiate from multiple sources, including the cardiopharyngeal mesoderm, which gives rise also to cardiac and branchiomeric muscles. The enhancers activated during endothelial differentiation within the cardiopharyngeal mesoderm are not completely known. Here, we use a cardiogenic mesoderm differentiation model that activates an endothelial transcription program to identify endothelial regulatory elements activated in early cardiogenic mesoderm. Integrating chromatin remodeling and gene expression data with available single-cell RNA-seq data from mouse embryos, we identify 101 putative regulatory elements of EC genes. We then apply a machine-learning strategy, trained on validated enhancers, to predict enhancers. Using this computational assay, we determine that 50% of these sequences are likely enhancers, some of which are already reported. We also identify a smaller set of regulatory elements of well-known EC genes and validate them using genetic and epigenetic perturbation. Finally, we integrate multiple data sources and computational tools to search for transcriptional factor binding motifs. In conclusion, we show EC regulatory sequences with a high likelihood to be enhancers, and we validate a subset of them using computational and cell culture models. Motif analyses show that the core EC transcription factors GATA/ETS/FOS is a likely driver of EC regulation in cardiopharyngeal mesoderm.

: The brain-related phenotypes observed in 22q11.2 deletion syndrome (DS) patients are highly variable, and their origin is poorly understood. Changes in brain metabolism might contribute to these phenotypes, as many of the deleted genes are involved in metabolic processes, but this is unknown. This study shows for the first time that Tbx1 haploinsufficiency causes brain metabolic imbalance. We studied two mouse models of 22q11.2DS using mass spectrometry, nuclear magnetic resonance spectroscopy, and transcriptomics. We found that Tbx1 +/- mice and Df1/+ mice, with a multigenic deletion that includes Tbx1, have elevated brain methylmalonic acid, which is highly brain-toxic. Focusing on Tbx1 mutants, we found that they also have a more general brain metabolomic imbalance that affects key metabolic pathways, such as glutamine-glutamate and fatty acid metabolism. We provide transcriptomic evidence of a genotype-vitamin B12 treatment interaction. In addition, vitamin B12 treatment rescued a behavioural anomaly in Tbx1 +/- mice. Further studies will be required to establish whether the specific metabolites affected by Tbx1 haploinsufficiency are potential biomarkers of brain disease status in 22q11.2DS patients.

Estimates networks of conditional dependencies (Gaussian graphical models) from multiple classes of data (similar but not exactly, i.e. measurements on different equipment, in different locations or for various sub-types). Package also allows to generate simulation data and evaluate the performance. Implementation of the method described in Angelini, De Canditiis and Plaksienko (2022) <doi:10.3390/math10213983>.

Estimates networks of conditional dependencies (Gaussian graphical models) from multiple classes of data (similar but not exactly, i.e. measurements on different equipment, in different locations or for various sub-types). Package also allows to generate simulation data and evaluate the performance. Implementation of the method described in Angelini, De Canditiis and Plaksienko (2022)

Bi-allelic hypomorphic mutations in DNMT3B disrupt DNA methyltransferase activity and lead to immunodeficiency, centromeric instability, facial anomalies syndrome, type 1 (ICF1). Although several ICF1 phenotypes have been linked to abnormally hypomethylated repetitive regions, the unique genomic regions responsible for the remaining disease phenotypes remain largely uncharacterized. Here we explored two ICF1 patient-derived induced pluripotent stem cells (iPSCs) and their CRISPR-Cas9-corrected clones to determine whether DNMT3B correction can globally overcome DNA methylation defects and related changes in the epigenome. Hypomethylated regions throughout the genome are highly comparable between ICF1 iPSCs carrying different DNMT3B variants, and significantly overlap with those in ICF1 patient peripheral blood and lymphoblastoid cell lines. These regions include large CpG island domains, as well as promoters and enhancers of several lineage-specific genes, in particular immune-related, suggesting that they are premarked during early development. CRISPR-corrected ICF1 iPSCs reveal that the majority of phenotype-related hypomethylated regions reacquire normal DNA methylation levels following editing. However, at the most severely hypomethylated regions in ICF1 iPSCs, which also display the highest increases in H3K4me3 levels and/or abnormal CTCF binding, the epigenetic memory persists, and hypomethylation remains uncorrected. Overall, we demonstrate that restoring the catalytic activity of DNMT3B can reverse the majority of the aberrant ICF1 epigenome. However, a small fraction of the genome is resilient to this rescue, highlighting the challenge of reverting disease states that are due to genome-wide epigenetic perturbations. Uncovering the basis for the persistent epigenetic memory will promote the development of strategies to overcome this obstacle.

CRC is an adult-onset carcinoma representing the third most common cancer and the second leading cause of cancer-related deaths in the world. EO-CRC (&lt;45 years of age) accounts for 5% of the CRC cases and is associated with cancer-predisposing genetic factors in half of them. Here, we describe the case of a woman affected by BWSp who developed EO-CRC at age 27. To look for a possible molecular link between BWSp and EO-CRC, we analysed her whole-genome genetic and epigenetic profiles in blood, and peri-neoplastic and neoplastic colon tissues. The results revealed a general instability of the tumor genome, including copy number and methylation changes affecting genes of the WNT signaling pathway, CRC biomarkers and imprinted loci. At the germline level, two missense mutations predicted to be likely pathogenic were found in compound heterozygosity affecting the Cystic Fibrosis (CF) gene CFTR that has been recently classified as a tumor suppressor gene, whose dysregulation represents a severe risk factor for developing CRC. We also detected constitutional loss of methylation of the KCNQ1OT1:TSS-DMR that leads to bi-allelic expression of the lncRNA KCNQ1OT1 and BWSp. Our results support the hypothesis that the inherited CFTR mutations, together with constitutional loss of methylation of the KCNQ1OT1:TSS-DMR, initiate the tumorigenesis process. Further somatic genetic and epigenetic changes enhancing the activation of the WNT/beta-catenin pathway likely contributed to increase the growth advantage of cancer cells. Although this study does not provide any conclusive cause-effect relationship between BWSp and CRC, it is tempting to speculate that the imprinting defect of BWSp might accelerate tumorigenesis in adult cancer in the presence of predisposing genetic variants.