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Background: Elderly individuals over 65, along with children under 5, are the most affected by invasive pneumococcal diseases (IPDs). Monitoring vaccination coverage and conducting surveillance are essential for guiding evidence-based prevention campaigns and public health measures. Methods: Since 2007, the Veneto Region has relied on three sources for surveillance of invasive bacterial infections, contributing to an increase in reported IPD cases. This study analyzed notifications related to individuals aged ≥65 years from 2007 to 2023. Results: A total of 1527 cases of IPDs in elderly individuals were reported between 2007 and 2023. The notification rate significantly increased from 5.61 to 14.63 per 100,000 inhabitants, despite underreporting during the COVID-19 pandemic. Cases associated with sepsis increased from 3.89 to 9.58 per 100,000, while notifications of meningitis and case fatality rates remained stable at 1.5 per 100,000 and 11.8%, respectively. Serotyping was not performed in 52% of the notifications. The most common serotypes were 3 (21.6%), 8 (11.1%), and 19A (5.0%), with fluctuations over time. There was a significant decline in serotypes covered by PCV7 and PCV13 and an increase in non-vaccine serotypes. Conclusions: The regional surveillance system allows for an increasingly comprehensive profile of the epidemiological landscape of IPDs in Veneto. However, the surveillance of pneumococcal infections still presents challenges. The currently available data are likely to be underestimated, mainly referring to the most severe cases, and the serotyping necessary to identify the etiological agent is still not often performed.

HPV is the most common cause of sexually-transmitted infections the world over. The aim of this study was to assess the impact of a healthcare quality improvement strategy designed to increase the rate of vaccination against HPV in women diagnosed with cervical lesions graded as CIN2 or higher (CIN2+) during routine screening. The Veneto Regional Health Service developed a 22-item questionnaire to measure the gap between ideal procedure and real practice regarding the offer of vaccination against HPV for women undergoing routine cervical screening. The questionnaire was administered to nine expert doctors, one at each of the region’s Local Health Units (LHUs). An additional specific assessment concerned the quality of the related web pages available on the LHU websites. Strategies to close the gap between ideal procedure and real practice were decided collegially, and a checklist to support good practices was developed and shared with operators at the LHUs. Changes in practice were measured using data relating to women diagnosed with CIN2+ lesions extracted from the regional oncological screening database before and after the publication of a Regional Procedure on the topic. The LHUs differed considerably in how they managed each step, in terms of training for healthcare personnel, organization and assessment of the pathway from cervical screening to HPV vaccination, and in dedicated website communication. After implementing the quality improvement strategy, the proportion of women given a first dose of HPV vaccine within 3 months of being diagnosed with CIN2+ lesions at 1st-level screening rose to 50% (compared with 30.85% beforehand), and the median time elapsing between a diagnosis of CIN2+ lesion and a first dose of HPV vaccine dropped from 158 to 90 days. These findings underscore the importance of providing training to promote vaccination for general practitioners and other clinicians. The study also confirms the need for more efforts in communication to ensure that any citizen has the opportunity to access preventive healthcare.

Abstract Background The epigenetic status of patients 6-month post-COVID-19 infection remains largely unexplored. The existence of long-COVID, or post-acute sequelae of SARS-CoV-2 infection (PASC), suggests potential long-term changes. Long-COVID includes symptoms like fatigue, neurological issues, and organ-related problems, regardless of initial infection severity. The mechanisms behind long-COVID are unclear, but virus-induced epigenetic changes could play a role. Methods and results Our study explores the lasting epigenetic impacts of SARS-CoV-2 infection. We analyzed genome-wide DNA methylation patterns in an Italian cohort of 96 patients 6 months after COVID-19 exposure, comparing them to 191 healthy controls. We identified 42 CpG sites with significant methylation differences (FDR

Abstract Background Individuals with type 2 diabetes (T2D) face an increased mortality risk, not fully captured by canonical risk factors. Biological age estimation through DNA methylation (DNAm), i.e. the epigenetic clocks, is emerging as a possible tool to improve risk stratification for multiple outcomes. However, whether these tools predict mortality independently of canonical risk factors in subjects with T2D is unknown. Methods Among a cohort of 568 T2D patients followed for 16.8 years, we selected a subgroup of 50 subjects, 27 survived and 23 deceased at present, passing the quality check and balanced for all risk factors after propensity score matching. We analyzed DNAm from peripheral blood leukocytes using the Infinium Human MethylationEPIC BeadChip (Illumina) to evaluate biological aging through previously validated epigenetic clocks and assess the DNAm-estimated levels of selected inflammatory proteins and blood cell counts. We tested the associations of these estimates with mortality using two-stage residual-outcome regression analysis, creating a reference model on data from the group of survived patients. Results Deceased subjects had higher median epigenetic age expressed with DNAmPhenoAge algorithm (57.49 [54.72; 60.58] years. vs. 53.40 [49.73; 56.75] years; p = 0.012), and accelerated DunedinPoAm pace of aging (1.05 [1.02; 1.11] vs. 1.02 [0.98; 1.06]; p = 0.012). DNAm PhenoAge (HR 1.16, 95% CI 1.05–1.28; p = 0.004) and DunedinPoAm (HR 3.65, 95% CI 1.43–9.35; p = 0.007) showed an association with mortality independently of canonical risk factors. The epigenetic predictors of 3 chronic inflammation-related proteins, i.e. CXCL10, CXCL11 and enRAGE, C-reactive protein methylation risk score and DNAm-based estimates of exhausted CD8 + T cell counts were higher in deceased subjects when compared to survived. Conclusions These findings suggest that biological aging, as estimated through existing epigenetic tools, is associated with mortality risk in individuals with T2D, independently of common risk factors and that increased DNAm-surrogates of inflammatory protein levels characterize deceased T2D patients. Replication in larger cohorts is needed to assess the potential of this approach to refine mortality risk in T2D.

Abstract Autism spectrum disorder (ASD) is a complex neurodevelopmental condition with a strong genetic component in which rare variants contribute significantly to risk. We performed whole genome and/or exome sequencing (WGS and WES) and SNP-array analysis to identify both rare sequence and copy number variants (SNVs and CNVs) in 435 individuals from 116 ASD families. We identified 37 rare potentially damaging de novo SNVs (pdSNVs) in the cases (n = 144). Interestingly, two of them (one stop-gain and one missense variant) occurred in the same gene, BRSK2. Moreover, the identification of 8 severe de novo pdSNVs in genes not previously implicated in ASD (AGPAT3, IRX5, MGAT5B, RAB8B, RAP1A, RASAL2, SLC9A1, YME1L1) highlighted promising candidates. Potentially damaging CNVs (pdCNVs) provided support to the involvement of inherited variants in PHF3, NEGR1, TIAM1 and HOMER1 in neurodevelopmental disorders (NDD), although mostly acting as susceptibility factors with incomplete penetrance. Interpretation of identified pdSNVs/pdCNVs according to the ACMG guidelines led to a molecular diagnosis in 19/144 cases, although this figure represents a lower limit and is expected to increase thanks to further clarification of the role of likely pathogenic variants in ASD/NDD candidate genes not yet established. In conclusion, our study highlights promising ASD candidate genes and contributes to characterize the allelic diversity, mode of inheritance and phenotypic impact of de novo and inherited risk variants in ASD/NDD genes.

Background/Objectives: HBV infections can lead to serious liver complications that can have fatal consequences. In 2022, around 1.1 million individuals died from HBV-related cirrhosis and hepatocellular carcinoma. Vaccines allow us to save more than 2.5 million lives each year; however, up to 10% of vaccinated individuals may not develop sufficient protective antibody levels. The aim of this study was to investigate the epigenetic drift in the response to HBV vaccine in isolated B cells. Methods: Epigenetic drift was measured by counting rare DNA methylation variants. These epivariants were detected in epigenome-wide data collected from isolated B cell samples from 41 responders and 30 non-responders (age range 22–62 years) to vaccination against HBV. Results: We found an accumulation of epivariants in the NR group, with a significant increase in hyper-methylated aberrations. We identified the chromosomes (1, 3, 11, 12, and 14) and genes (e.g., RUSC1_AS1 or TROVE2) particularly enriched in epivariants in NRs. The literature search and pathway analysis indicate that such genes are involved in the correct functioning of the immune system. Moreover, we observed a correlation between epigenetic drift and DNA methylation entropy in the male population of the cohort. Finally, we confirmed the correlation between epivariant loads and age-related epigenetic clocks. Conclusions: Our findings support the idea that an age-related derangement of the epigenetic architecture is involved in unresponsiveness to the HBV vaccine. Furthermore, the overall results highlight the interconnection between various epigenetic dynamics (such as drift, clocks, and entropy), although these interconnections seem not to be involved in the altered immunological activity.

Background/Objectives: Major depressive disorder (MDD) is a mental health condition that can severely impact patients’ social lives, leading to withdrawal and difficulty in maintaining relationships. Environmental factors such as trauma and stress can worsen MDD by interacting with genetic predispositions. Epigenetics, which examines changes in gene expression influenced by the environment, may help identify patterns linked to depression. This study aimed to explore the epigenetic mechanisms behind MDD by analysing six public datasets (n = 1125 MDD cases, 398 controls in blood; n = 95 MDD cases, 96 controls in brain tissues) from the Gene Expression Omnibus. Methods: As an innovative approach, two meta-analyses of DNA methylation patterns were conducted alongside an investigation of stochastic epigenetic mutations (SEMs), epigenetic age acceleration, and rare epivariations. Results: While no significant global methylation differences were observed between MDD cases and controls, hypomethylation near the SHF gene (brain-specific probe cg25801113) was consistently found in MDD cases. SEMs revealed a gene-level burden in MDD, though epigenetic age acceleration was not central to the disorder. Additionally, 51 rare epivariations were identified in blood tissue and 1 in brain tissue linked to MDD. Conclusions: The study emphasises the potential role of rare epivariations in MDD’s epigenetic regulation but calls for further research with larger, more diverse cohorts to confirm these findings.

Healthcare workers (HCWs) are a high-risk group for hepatitis B virus (HBV) infection. Notably, about 5–10% of the general population does not respond to the HBV vaccination. In this study, we aimed to investigate DNA methylation (DNAm) in order to estimate the biological age of B cells from HCW of both sexes, either responder (R) or non-responder (NR), to HBV vaccination. We used genome-wide DNA methylation data to calculate a set of biomarkers in B cells collected from 41 Rs and 30 NRs between 22 and 62 years old. Unresponsiveness to HBV vaccination was associated with accelerated epigenetic aging (DNAmAge, AltumAge, DunedinPoAm) and was accompanied by epigenetic drift. Female non-responders had higher estimates of telomere length and lower CRP inflammation risk score when compared to responders. Overall, epigenetic differences between responders and non-responders were more evident in females than males. In this study we demonstrated that several methylation DNAm-based clocks and biomarkers are associated with an increased risk of non-response to HBV vaccination, particularly in females. Based on these results, we propose that accelerated epigenetic age could contribute to vaccine unresponsiveness. These insights may help improve the evaluation of the effectiveness of vaccination strategies, especially among HCWs and vulnerable patients.

Abstract Isolated REM Sleep Behavior Disorder (iRBD) is the strongest prodromal marker for α-synucleinopathies. Overt α-synucleinopathies and aging share several mechanisms, but this relationship has been poorly investigated in prodromal phases. Using DNA methylation-based epigenetic clocks, we measured biological aging in videopolysomnography confirmed iRBD patients, videopolysomnography-negative and population-based controls. We found that iRBDs tended to be epigenetically older than controls, suggesting that accelerated aging characterizes prodromal neurodegeneration.

Abstract Background COVID-19 has a wide spectrum of clinical manifestations and given its impact on morbidity and mortality, there is an unmet medical need to discover endogenous cellular and molecular biomarkers that predict the expected clinical course of the disease. Recently, epigenetics and especially DNA methylation have been pointed out as a promising tool for outcome prediction in several diseases. Methods and results Using the Illumina Infinium Methylation EPIC BeadChip850K, we investigated genome-wide differences in DNA methylation in an Italian Cohort of patients with comorbidities and compared severe (n = 64) and mild (123) prognosis. Results showed that the epigenetic signature, already present at the time of Hospital admission, can significantly predict risk of severe outcomes. Further analyses provided evidence of an association between age acceleration and a severe prognosis after COVID-19 infection. The burden of Stochastic Epigenetic Mutation (SEMs) has been significantly increased in patients with poor prognosis. Results have been replicated in silico considering COVID-19 negative subjects and available previously published datasets. Conclusions Using original methylation data and taking advantage of already published datasets, we confirmed in the blood that epigenetics is actively involved in immune response after COVID-19 infection, allowing the identification of a specific signature able to discriminate the disease evolution. Furthermore, the study showed that epigenetic drift and age acceleration are associated with severe prognosis. All these findings prove that host epigenetics undergoes notable and specific rearrangements to respond to COVID-19 infection which can be used for a personalized, timely, and targeted management of COVID-19 patients during the first stages of hospitalization.

During the last decades, our knowledge about the genetic architecture of sporadic amyotrophic lateral sclerosis (sALS) has significantly increased. However, besides the recognized genetic risk factors, also the environment is supposed to have a role in disease pathogenesis. Epigenetic modifications reflect the results of the interaction between environmental factors and genes and may play a role in the development and progression of ALS. A recent epigenome-wide association study (EWAS) in blood identified differentially methylated positions mapping to 42 genes involved in cholesterol biosynthesis and immune-related pathways. Here we performed a genome-wide DNA methylation analysis in the blood of an Italian cohort of 61 sALS patients and 61 healthy controls. Initially, a conventional genome-wide association analysis was performed, and results were subsequently integrated with the findings from the previous EWAS using a meta-analytical approach. To delve deeper into the significant outcomes, over-representation analysis (ORA) was employed. Moreover, the epigenetic signature obtained from the meta-analysis was examined to determine potential associations with chemical compounds, utilizing the Toxicogenomic Database. Expanding the scope of the epigenetic analysis, we explored both epigenetic drift and rare epivariations. Notably, we observed an elevated epigenetic drift in sALS patients compared to controls, both at a global and single gene level. Interestingly, epigenetic drift at a single gene level revealed an enrichment of genes related to the neurotrophin signaling pathway. Moreover, for the first time, we identified rare epivariations exclusively enriched in sALS cases associated with 153 genes, 88 of whom with a strong expression in cerebral areas. Overall, our study reinforces the evidence that epigenetics may contribute to the pathogenesis of ALS and that epigenetic drift may be a useful diagnostic marker. Moreover, this study suggests the potential role of epivariations in ALS.

Background Sudden infant death syndrome (SIDS) is the leading cause of death up to age 1. Sudden unexplained death in childhood (SUDC) is similar but affects mostly toddlers aged 1 to 4. SUDC is rarer than SIDS, and although cardiogenetic testing (molecular autopsy) identifies an underlying cause in a fraction of SIDS, less is known about SUDC. Methods and Results Seventy‐seven SIDS and 16 SUDC cases underwent molecular autopsy with 25 definitive‐evidence arrhythmia‐associated genes. In 18 cases, another 76 genes with varying degrees of evidence were analyzed. Parents were offered cascade screening. Double‐blind review of clinical‐genetic data established genotype–phenotype correlations. The yield of likely pathogenic variants in the 25 genes was higher in SUDC than in SIDS (18.8% [3/16] versus 2.6% [2/77], respectively; P=0.03), whereas novel/ultra‐rare variants of uncertain significance were comparably represented. Rare variants of uncertain significance and likely benign variants were found only in SIDS. In cases with expanded analyses, likely pathogenic/likely benign variants stemmed only from definitive‐evidence genes, whereas all other genes contributed only variants of uncertain significance. Among 24 parents screened, variant status and phenotype largely agreed, and 3 cases positively correlated for cardiac channelopathies. Genotype–phenotype correlations significantly aided variant adjudication. Conclusions Genetic yield is higher in SUDC than in SIDS although, in both, it is contributed only by definitive‐evidence genes. SIDS/SUDC cascade family screening facilitates establishment or dismissal of a diagnosis through definitive variant adjudication indicating that anonymity is no longer justifiable. Channelopathies may underlie a relevant fraction of SUDC. Binary classifications of genetic causality (pathogenic versus benign) could not always be adequate.

Introduction Prevention of cardiovascular disease (CVD) is of key importance in reducing morbidity, disability and mortality worldwide. Observational studies suggest that digital health interventions can be an effective strategy to reduce cardiovascular (CV) risk. However, evidence from large randomised clinical trials is lacking.Methods and analysis The CV-PREVITAL study is a multicentre, prospective, randomised, controlled, open-label interventional trial designed to compare the effectiveness of an educational and motivational mobile health (mHealth) intervention versus usual care in reducing CV risk. The intervention aims at improving diet, physical activity, sleep quality, psycho-behavioural aspects, as well as promoting smoking cessation and adherence to pharmacological treatment for CV risk factors. The trial aims to enrol approximately 80 000 subjects without overt CVDs referring to general practitioners’ offices, community pharmacies or clinics of Scientific Institute for Research, Hospitalization and Health Care (Italian acronym IRCCS) affiliated with the Italian Cardiology Network. All participants are evaluated at baseline and after 12 months to assess the effectiveness of the intervention on short-term endpoints, namely improvement in CV risk score and reduction of major CV risk factors. Beyond the funded life of the study, a long-term (7 years) follow-up is also planned to assess the effectiveness of the intervention on the incidence of major adverse CV events. A series of ancillary studies designed to evaluate the effect of the mHealth intervention on additional risk biomarkers are also performed.Ethics and dissemination This study received ethics approval from the ethics committee of the coordinating centre (Monzino Cardiology Center; R1256/20-CCM 1319) and from all other relevant IRBs and ethics committees. Findings are disseminated through scientific meetings and peer-reviewed journals and via social media. Partners are informed about the study’s course and findings through regular meetings.Trial registration number NCT05339841.

IntroductionFOXE1 is required for thyroid function and its homozygous mutations cause a rare syndromic form of congenital hypothyroidism (CH). FOXE1 has a polymorphic polyalanine tract whose involvement in thyroid pathology is controversial. Starting from genetic studies in a CH family, we explored the functional role and involvement of FOXE1 variations in a large CH population.MethodsWe applied NGS screening to a large CH family and a cohort of 1752 individuals and validated these results by in silico modeling and in vitro experiments.ResultsA new heterozygous FOXE1 variant segregated with 14-Alanine tract homozygosity in 5 CH siblings with athyreosis. The p.L107V variant demonstrated to significantly reduce the FOXE1 transcriptional activity. The 14-Alanine-FOXE1 displayed altered subcellular localization and significantly impaired synergy with other transcription factors, when compared with the more common 16-Alanine-FOXE1. The CH group with thyroid dysgenesis was largely and significantly enriched with the 14-Alanine-FOXE1 homozygosity.DiscussionWe provide new evidence that disentangle the pathophysiological role of FOXE1 polyalanine tract, thereby significantly broadening the perspective on the role of FOXE1 in the complex pathogenesis of CH. FOXE1 should be therefore added to the group of polyalanine disease-associated transcription factors.

BackgroundThe UNC13A gene is an established susceptibility locus for amyotrophic lateral sclerosis (ALS) and a determinant of shorter survival after disease onset, with up to 33.0 months difference in life expectancy for carriers of the rs12608932 risk genotype. However, its overall effect on other clinical features and ALS phenotypic variability is controversial.MethodsGenotype data of the UNC13A rs12608932 SNP (A–major allele; C–minor allele) was obtained from a cohort of 972 ALS patients. Demographic and clinical variables were collected, including cognitive and behavioral profiles, evaluated through the Edinburgh Cognitive and Behavioral ALS Screen (ECAS) – Italian version and the Frontal Behavioral Inventory (FBI); upper and lower motor neuron involvement, assessed by the Penn Upper Motor Neuron Score (PUMNS) and the Lower Motor Neuron Score (LMNS)/Medical Research Council (MRC) scores, respectively; the ALS Functional Rating Scale Revised (ALSFRS-R) score at evaluation and progression rate; age and site of onset; survival. The comparison between the three rs12608932 genotypes (AA, AC, and CC) was performed using the additive, dominant, and recessive genetic models.ResultsThe rs12608932 minor allele frequency was 0.31 in our ALS cohort, in comparison to 0.33–0.41 reported in other Caucasian ALS populations. Carriers of at least one minor C allele (AC + CC genotypes) had a shorter median survival than patients with the wild-type AA genotype (−11.7 months, p = 0.013), even after adjusting for age and site of onset, C9orf72 mutational status and gender. Patients harboring at least one major A allele (AA + AC genotypes) and particularly those with the wild-type AA genotype showed a significantly higher PUMNS compared to CC carriers (p = 0.015 and padj = 0.037, respectively), thus indicating a more severe upper motor neuron involvement. Our analysis did not detect significant associations with all the other clinical parameters considered.ConclusionOverall, our findings confirm the role of UNC13A as a determinant of survival in ALS patients and show the association of this locus also with upper motor neuron involvement.

Background: Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease affecting upper and/or lower motor neurons and characterized by complex etiology. Familial cases show high genetic heterogeneity and sporadic cases (90%) are associated with several genetic and environmental risk factors. Among the genetic risk factors, the contribution of non-coding elements, such as microRNAs (miRNAs), to ALS disease susceptibility remains largely unexplored.Aim: This work aims to identify rare variants in miRNA genes in sporadic ALS (sALS) patients which may cause a defective miRNA maturation or altered target gene recognition by changing miRNA secondary structure or seed sequence, respectively.Methods: Rare variants located in miRNA loci with a minor allele frequency (MAF) < 0.01 were extracted from whole genome sequencing (WGS) data of 100 sALS patients. The secondary pre-miRNA structures were predicted using MiRVas to evaluate the impact of the variants on RNA folding process. Human TargetScan was used to retrieve all the potential target genes of miRNAs with variants in the seed region. Over Representation Analysis (ORA) was conducted to compare the lists of target genes for the reference and mutated miRNAs in the seed sequence.Results: Our analysis identified 86 rare variants in 77 distinct miRNAs and distributed in different parts of the miRNA precursors. The presence of these variants changed miRNA secondary structures in ∼70% of MiRVas predictions. By focusing on the 6 rare variants mapping within the seed sequence, the predicted target genes increased in number compared to the reference miRNA and included novel targets in a proportion ranging from 30 to 82%. Interestingly, ORA revealed significant changes in gene set enrichment only for mutated miR-509-1 and miR-941-3 for which the Gene Ontology term related to “nervous system development” was absent and present, respectively, compared to target lists of the reference miRNA.Conclusion: We here developed a workflow to study miRNA rare variants from WGS data and to predict their biological effects on miRNA folding, maturation and target gene recognition. Although this in silico approach certainly needs functional validation in vitro and in vivo, it may help define the role of miRNA variability in ALS and complex diseases.

Multiple Sclerosis (MS) is a complex multifactorial autoimmune disease, whose sex- and age-adjusted prevalence in Sardinia (Italy) is among the highest worldwide. To date, 233 loci were associated with MS and almost 20% of risk heritability is attributable to common genetic variants, but many low-frequency and rare variants remain to be discovered. Here, we aimed to contribute to the understanding of the genetic basis of MS by investigating potentially functional rare variants. To this end, we analyzed thirteen multiplex Sardinian families with Immunochip genotyping data. For five families, Whole Exome Sequencing (WES) data were also available. Firstly, we performed a non-parametric Homozygosity Haplotype analysis for identifying the Region from Common Ancestor (RCA). Then, on these potential disease-linked RCA, we searched for the presence of rare variants shared by the affected individuals by analyzing WES data. We found: (i) a variant (43181034 T > G) in the splicing region on exon 27 of CUL9; (ii) a variant (50245517 A > C) in the splicing region on exon 16 of ATP9A; (iii) a non-synonymous variant (43223539 A > C), on exon 9 of TTBK1; (iv) a non-synonymous variant (42976917 A > C) on exon 9 of PPP2R5D; and v) a variant (109859349-109859354) in 3′UTR of MYO16.

Marfan syndrome (MFS) is a rare inherited autosomic disorder, which encompasses a variety of systemic manifestations caused by mutations in the Fibrillin-1 encoding gene (FBN1). Cardinal clinical phenotypes of MFS are highly variable in terms of severity, and commonly involve cardiovascular, ocular, and musculoskeletal systems with a wide range of manifestations, such as ascending aorta aneurysms and dissection, mitral valve prolapse, ectopia lentis and long bone overgrowth, respectively. Of note, an accurate and prompt diagnosis is pivotal in order to provide the best treatment to the patients as early as possible. To date, the diagnosis of the syndrome has relied upon a systemic score calculation as well as DNA mutation identification. The aim of this review is to summarize the latest MFS evidence regarding the definition, differences and similarities with other connective tissue pathologies with severe systemic phenotypes (e.g., Autosomal dominant Weill–Marchesani syndrome, Loeys–Dietz syndrome, Ehlers–Danlos syndrome) and clinical assessment. In this regard, the management of MFS requires a multidisciplinary team in order to accurately control the evolution of the most severe and potentially life-threatening complications. Based on recent findings in the literature and our clinical experience, we propose a multidisciplinary approach involving specialists in different clinical fields (i.e., cardiologists, surgeons, ophthalmologists, orthopedics, pneumologists, neurologists, endocrinologists, geneticists, and psychologists) to comprehensively characterize, treat, and manage MFS patients with a personalized medicine approach.

Abstract Calabrian Greeks are an enigmatic population that have preserved and evolved a unique variety of language, Greco, survived in the isolated Aspromonte mountain area of Southern Italy. To understand their genetic ancestry and explore possible effects of geographic and cultural isolation, we genome-wide genotyped a large set of South Italian samples including both communities that still speak Greco nowadays and those that lost the use of this language earlier in time. Comparisons with modern and ancient populations highlighted ancient, long-lasting genetic links with Eastern Mediterranean and Caucasian/Near-Eastern groups as ancestral sources of Southern Italians. Our results suggest that the Aspromonte communities might be interpreted as genetically drifted remnants that departed from such ancient genetic background as a consequence of long-term isolation. Specific patterns of population structuring and higher levels of genetic drift were indeed observed in these populations, reflecting geographic isolation amplified by cultural differences in the groups that still conserve the Greco language. Isolation and drift also affected the current genetic differentiation at specific gene pathways, prompting for future genome-wide association studies aimed at exploring trait-related loci that have drifted up in frequency in these isolated groups.

Abstract Adoption of diets based on some cereals, especially on rice, signified an iconic change in nutritional habits for many Asian populations and a relevant challenge for their capability to maintain glucose homeostasis. Indeed, rice shows the highest carbohydrates content and glycemic index among the domesticated cereals and its usual ingestion represents a potential risk factor for developing insulin resistance and related metabolic diseases. Nevertheless, type 2 diabetes and obesity epidemiological patterns differ among Asian populations that rely on rice as a staple food, with higher diabetes prevalence and increased levels of central adiposity observed in people of South Asian ancestry rather than in East Asians. This may be at least partly due to the fact that populations from East Asian regions where wild rice or other cereals such as millet have been already consumed before their cultivation and/or were early domesticated have relied on these nutritional resources for a period long enough to have possibly evolved biological adaptations that counteract their detrimental side effects. To test such a hypothesis, we compared adaptive evolution of these populations with that of control groups from regions where the adoption of cereal‐based diets occurred many thousand years later and which were identified from a genome‐wide dataset including 2,379 individuals from 124 East Asian and South Asian populations. This revealed selective sweeps and polygenic adaptive mechanisms affecting functional pathways involved in fatty acids metabolism, cholesterol/triglycerides biosynthesis from carbohydrates, regulation of glucose homeostasis, and production of retinoic acid in Chinese Han and Tujia ethnic groups, as well as in people of Korean and Japanese ancestry. Accordingly, long‐standing rice‐ and/or millet‐based diets have possibly contributed to trigger the evolution of such biological adaptations, which might represent one of the factors that play a role in mitigating the metabolic risk of these East Asian populations.

The aim of the present study was to provide a comprehensive characterization of whole genome DNA methylation patterns in replicative and ionizing irradiation- or doxorubicin-induced premature senescence, exhaustively exploring epigenetic modifications in three different human cell types: in somatic diploid skin fibroblasts and in bone marrow- and adipose-derived mesenchymal stem cells. With CpG-wise differential analysis, three epigenetic signatures were identified: (a) cell type- and treatment-specific signature; (b) cell type-specific senescence-related signature; and (c) cell type-transversal replicative senescence-related signature. Cluster analysis revealed that only replicative senescent cells created a distinct group reflecting notable alterations in the DNA methylation patterns accompanying this cellular state. Replicative senescence-associated epigenetic changes seemed to be of such an extent that they surpassed interpersonal dissimilarities. Enrichment in pathways linked to the nervous system and involved in the neurological functions was shown after pathway analysis of genes involved in the cell type-transversal replicative senescence-related signature. Although DNA methylation clock analysis provided no statistically significant evidence on epigenetic age acceleration related to senescence, a persistent trend of increased biological age in replicative senescent cultures of all three cell types was observed. Overall, this work indicates the heterogeneity of senescent cells depending on the tissue of origin and the type of senescence inducer that could be putatively translated to a distinct impact on tissue homeostasis.

Primary ovarian insufficiency (POI) is one of the major causes of female infertility associated with the premature loss of ovarian function in about 3.7% of women before the age of 40. This disorder is highly heterogeneous and can manifest with a wide range of clinical phenotypes, ranging from ovarian dysgenesis and primary amenorrhea to post-pubertal secondary amenorrhea, with elevated serum gonadotropins and hypoestrogenism. The ovarian defect still remains idiopathic in some cases; however, a strong genetic component has been demonstrated by the next-generation sequencing (NGS) approach of familiar and sporadic POI cases. As recent evidence suggested an oligogenic architecture for POI, we developed a target NGS panel with 295 genes including known candidates and novel genetic determinants potentially involved in POI pathogenesis. Sixty-four patients with early onset POI (range: 10–25 years) of our cohort have been screened with 90% of target coverage at 50×. Here, we report 48 analyzed patients with at least one genetic variant (75%) in the selected candidate genes. In particular, we found the following: 11/64 patients (17%) with two variants, 9/64 (14%) with three variants, 9/64 (14%) with four variants, 3/64 (5%) with five variants, and 2/64 (3%) with six variants. The most severe phenotypes were associated with either the major number of variations or a worse prediction in pathogenicity of variants. Bioinformatic gene ontology analysis identified the following major pathways likely affected by gene variants: 1) cell cycle, meiosis, and DNA repair; 2) extracellular matrix remodeling; 3) reproduction; 4) cell metabolism; 5) cell proliferation; 6) calcium homeostasis; 7) NOTCH signaling; 8) signal transduction; 9) WNT signaling; 10) cell death; and 11) ubiquitin modifications. Consistently, the identified pathways have been described in other studies dissecting the mechanisms of folliculogenesis in animal models of altered fertility. In conclusion, our results contribute to define POI as an oligogenic disease and suggest novel candidates to be investigated in patients with POI.

Multiple Sclerosis (MS) exhibits considerable heterogeneity in phenotypic expression, course, prognosis and response to therapy. This suggests this disease involves multiple, as yet poorly understood, causal mechanisms. In this work we assessed the possible causal link between gene expression level of five selected genes related to the pro-inflammatory NF-κB signaling pathway (i.e., CCL2, NFKB1, MAPK14, TNFRSF1A, CXCL10) in ten different brain tissues (i.e., cerebellum, frontal cortex, hippocampus, medulla, occipital cortex, putamen, substantia nigra, thalamus, temporal cortex and intralobular white matter) and MS. We adopted a two-stage Mendelian Randomization (MR) approach for the estimation of the causal effects of interest, based on summary-level data from 20 multiplex Sardinian families and data provided by the United Kingdom Brain Expression Consortium (UKBEC). Through Radial-MR and Cochrane’s Q statistics we identified and removed genetic variants which are most likely to be invalid instruments. To estimate the total causal effect, univariable MR was carried out separately for each gene and brain region. We used Inverse-Variance Weighted estimator (IVW) as main analysis and MR-Egger Regression estimator (MR-ER) and Weighted Median Estimator (WME) as sensitivity analysis. As these genes belong to the same pathway and thus they can be closely related, we also estimated their direct causal effects by applying IVW and MR-ER within a multivariable MR (MVMR) approach using set of genetic instruments specific and common (composite) to each multiple exposures represented by the expression of the candidate genes. Univariate MR analysis showed a significant positive total causal effect for CCL2 and NFKB1 respectively in medulla and cerebellum. MVMR showed a direct positive causal effect for NFKB1 and TNFRSF1A, and a direct negative causal effect for CCL2 in cerebellum; while in medulla we observed a direct positive causal effect for CCL2. Since in general we observed a different magnitude for the gene specific causal effect we hypothesize that in cerebellum and medulla the effect of each gene expression is direct but also mediated by the others. These results confirm the importance of the involvement of NF-κB signaling pathway in brain tissue for the development of the disease and improve our understanding in the pathogenesis of MS.

Baller-Gerold (BGS, MIM#218600) and Roberts (RBS, MIM#268300) syndromes are rare autosomal recessive disorders caused, respectively, by biallelic alterations in RECQL4 (MIM*603780) and ESCO2 (MIM*609353) genes. Common features are severe growth retardation, limbs shortening and craniofacial abnormalities which may include craniosynostosis. We aimed at unveiling the genetic lesions underpinning the phenotype of two unrelated children with a presumptive BGS diagnosis: patient 1 is a Turkish girl with short stature, microcephaly, craniosynostosis, seizures, intellectual disability, midface hemangioma, bilateral radial and thumb aplasia, tibial hypoplasia, and pes equinovarus. Patient 2 is an Iranian girl born to consanguineous parents with craniosynostosis, micrognathism, bilateral radial aplasia, thumbs, and foot deformity in the context of developmental delay. Upon negative RECQL4 test, whole exome sequencing (WES) analysis performed on the two trios led to the identification of two different ESCO2 homozygous inactivating variants: a previously described c.1131+1G>A transition in patient 1 and an unreported deletion, c.417del, in patient 2, thus turning the diagnosis into Roberts syndrome. The occurrence of a Baller-Gerold phenotype in two unrelated patients that were ultimately diagnosed with RBS demonstrates the strength of WES in redefining the nosological landscape of rare congenital malformation syndromes, a premise to yield optimized patients management and family counseling.

Gliomas are the most common primary neoplasm of the central nervous system. A promising frontier in the definition of glioma prognosis and treatment is represented by epigenetics. Furthermore, in this study, we developed a machine learning classification model based on epigenetic data (CpG probes) to separate patients according to their state of immunosuppression. We considered 573 cases of low-grade glioma (LGG) and glioblastoma (GBM) from The Cancer Genome Atlas (TCGA). First, from gene expression data, we derived a novel binary indicator to flag patients with a favorable immune state. Then, based on previous studies, we selected the genes related to the immune state of tumor microenvironment. After, we improved the selection with a data-driven procedure, based on Boruta. Finally, we tuned, trained, and evaluated both random forest and neural network classifiers on the resulting dataset. We found that a multi-layer perceptron network fed by the 338 probes selected by applying both expert choice and Boruta results in the best performance, achieving an out-of-sample accuracy of 82.8%, a Matthews correlation coefficient of 0.657, and an area under the ROC curve of 0.9. Based on the proposed model, we provided a method to stratify glioma patients according to their epigenomic state.

The diagnosis of Marfan spectrum includes a large number of clinical criteria. Although the identification of pathogenic variants contributes to the diagnostic process, its value to the prediction of clinical outcomes is still limited. An important novelty of the present study is represented by the statistical approach adopted to investigate genotype-phenotype correlation. The analysis has been improved considering the extended genetic information obtained by Next Generation Sequencing (NGS) and combining the effects of both rare and common genetic variants in an inclusive model. To this aim a cohort of 181 patients were analyzed with a NGS panel including 11 genes associated with Marfan spectrum. The genotype-phenotype correlation was also investigated considering the possibility to predict presence of a pathological mutation in Marfan syndrome (MFS) main genes based only on the analysis of phenotypic traits. Results obtained indicate that information about clinical traits can be summarized in a new variable that resulted significantly associated with the probability to find a pathological mutation in MFS main genes. This is important since the choice of the genetic test is often influenced by the phenotypic characterization of patients. Moreover, both rare and common variants were found to significantly contribute to clinical spectrum and their combination allowed to increase the percentage of phenotype variability that could be explained based on genetic factors. Results highlight the opportunity to take advantage of the overall genetic information obtained by NGS data to have a better clinical classification of patients.

Crohn's disease (CD) is notably characterized by the expansion of visceral fat with small adipocytes expressing a high proportion of anti-inflammatory genes. Conversely, visceral fat depots in ulcerative colitis (UC) patients have never been characterized. Our study aims were a) to compare adipocyte morphology and gene expression profile and bacterial translocation in omental (OM) and mesenteric (MES) adipose tissue of patients with UC and CD, and b) to investigate the effect of bacterial infection on adipocyte proliferation in vitro. Specimens of OM and MES were collected from 11 UC and 11 CD patients, processed and examined by light microscopy. Gene expression profiles were evaluated in adipocytes isolated from visceral adipose tissue using microarray and RTqPCR validations. Bacteria within adipose tissue were immuno-detected by confocal scanning laser microscopy. Adipocytes were incubated with Enterococcus faecalis and cells counted after 24 h. Morphology and molecular profile of OM and MES revealed that UC adipose tissue is less inflamed than CD adipose tissue. Genes linked to inflammation, bacterial response, chemotaxis and angiogenesis were down-regulated in adipocytes from UC compared to CD, whereas genes related to metallothioneins, apoptosis pathways and growth factor binding were up-regulated. A dense perinuclear positivity for Enterococcus faecalis was detected in visceral adipocytes from CD, whereas positivity was weak in UC. In vitro bacterial infection was associated with a five-fold increase in the proliferation rate of OM preadipocytes. Compared to UC, visceral adipose tissue from CD is more inflamed and more colonized by intestinal bacteria, which increase adipocyte proliferation. The influence of bacteria stored within adipocytes on the clinical course of IBD warrants further investigations.

Hypertension is a heritable and major contributor to the global burden of disease. The sum of rare and common genetic variants robustly identified so far explain only 1%-2% of the population variation in BP and hypertension. This suggests the existence of more undiscovered common variants. We conducted a genome-wide association study in 1,621 hypertensive cases and 1,699 controls and follow-up validation analyses in 19,845 cases and 16,541 controls using an extreme case-control design. We identified a locus on chromosome 16 in the 5' region of Uromodulin (UMOD; rs13333226, combined P value of 3.6 × 10⁻¹¹). The minor G allele is associated with a lower risk of hypertension (OR [95%CI]: 0.87 [0.84-0.91]), reduced urinary uromodulin excretion, better renal function; and each copy of the G allele is associated with a 7.7% reduction in risk of CVD events after adjusting for age, sex, BMI, and smoking status (H.R. = 0.923, 95% CI 0.860-0.991; p = 0.027). In a subset of 13,446 individuals with estimated glomerular filtration rate (eGFR) measurements, we show that rs13333226 is independently associated with hypertension (unadjusted for eGFR: 0.89 [0.83-0.96], p = 0.004; after eGFR adjustment: 0.89 [0.83-0.96], p = 0.003). In clinical functional studies, we also consistently show the minor G allele is associated with lower urinary uromodulin excretion. The exclusive expression of uromodulin in the thick portion of the ascending limb of Henle suggests a putative role of this variant in hypertension through an effect on sodium homeostasis. The newly discovered UMOD locus for hypertension has the potential to give new insights into the role of uromodulin in BP regulation and to identify novel drugable targets for reducing cardiovascular risk.