Synthetic eco-evolutionary dynamics in simple molecular environment.

The understanding of eco-evolutionary dynamics, and in particular the mechanism of coexistence of species, is still fragmentary and in need of test bench model systems. To this aim we developed a variant of SELEX in vitro selection to study the evolution of a population of ∼1015 single-strand DNA oligonucleotide 'individuals'. We begin with a seed of random sequences which we select via affinity capture from ∼1012 DNA oligomers of fixed sequence ('resources') over which they compete. At each cycle ('generation'), the ecosystem is replenished via PCR amplification of survivors. Massive parallel sequencing indicates that across generations the variety of sequences ('species') drastically decreases, while some of them become populous and dominate the ecosystem. The simplicity of our approach, in which survival is granted by hybridization, enables a quantitative investigation of fitness through a statistical analysis of binding energies. We find that the strength of individual resource binding dominates the selection in the first generations, while inter- and intra-individual interactions become important in later stages, in parallel with the emergence of prototypical forms of mutualism and parasitism.

Genetic susceptibility to severe COVID-19.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the etiologic agent of the coronavirus disease 2019 (COVID-19) pandemic. Clinical manifestations of the disease range from an asymptomatic condition to life-threatening events and death, with more severe courses being associated with age, male sex, and comorbidities. Besides these risk factors, intrinsic characteristics of the virus as well as genetic factors of the host are expected to account for COVID-19 clinical heterogeneity. Genetic studies have long been recognized as fundamental to identify biological mechanisms underlying congenital diseases, to pinpoint genes/proteins responsible for the susceptibility to different inherited conditions, to highlight targets of therapeutic relevance, to suggest drug repurposing, and even to clarify causal relationships that make modifiable some environmental risk factors. Though these studies usually take long time to be concluded and, above all, to translate their discoveries to patients' bedside, the scientific community moved really fast to deliver genetic signals underlying different COVID-19 phenotypes. In this Review, besides a concise description of COVID-19 symptomatology and of SARS-CoV-2 mechanism of infection, we aimed to recapitulate the current literature in terms of host genetic factors that specifically associate with an increased severity of the disease.

The circular RNA landscape in multiple sclerosis: Disease-specific associated variants and exon methylation shape circular RNA expression profile.

BACKGROUND: Circular RNAs (circRNAs) are a class of non-coding RNAs increasingly emerging as crucial actors in the pathogenesis of human diseases, including autoimmune and neurological disorders as multiple sclerosis (MS). Despite several efforts, the mechanisms regulating circRNAs expression are still largely unknown and the circRNA profile and regulation in MS-relevant cell models has not been completely investigated. In this work, we aimed at exploring the global landscape of circRNA expression in MS patients, also evaluating a possible correlation with their genetic and epigenetic background. METHODS: We performed RNA-seq experiments on circRNA-enriched samples, derived from peripheral blood mononuclear cells (PBMCs) of 10 MS patients and 10 matched controls and performed differential circRNA expression. The genetic background was evaluated using array genotyping, and an expression quantitative trait loci (eQTL) analysis was carried out. RESULTS: Expression analysis revealed 166 differentially expressed circRNAs in MS patients, 125 of which are downregulated. One of the top dysregulated circRNAs, hsa_circ_0007990, derives from the PGAP3 gene, encoding a protein relevant for the control of autoimmune responses. The downregulation of this circRNA was confirmed in two independent replication cohorts, suggesting its implementation as a possible RNA-based biomarker. The eQTL analysis evidenced a significant association between 89 MS-associated loci and the expression of at least one circRNA, suggesting that MS-associated variants could impact on disease pathogenesis by altering circRNA profiles. Finally, we found a significant correlation between exon methylation and circRNA expression levels, supporting the hypothesis that epigenetic features may play an important role in the definition of the cell circRNA pool. CONCLUSION: We described the circRNA expression profile of PBMCs in MS patients, suggesting that MS-associated variants may tune the expression levels of circRNAs acting as "circ-QTLs", and proposing a role for exon-based DNA methylation in regulating circRNA expression.

Sex-Related Differences in Long-Term Outcomes After Early-Onset Myocardial Infarction.

Importance: There is growing awareness of sex-related differences in cardiovascular risk profiles, but less is known about whether these extend to pre-menopausal females experiencing an early-onset myocardial infarction (MI), who may benefit from the protective effects of estrogen exposure. Methods: A nationwide study involving 125 Italian Coronary Care Units recruited 2,000 patients between 1998 and 2002 hospitalized for a type I myocardial infarction before the age of 45 years (male, n = 1,778 (88.9%). Patients were followed up for a median of 19.9 years (IQR 18.1-22.6). The primary composite endpoint was the occurrence of cardiovascular death, non-fatal myocardial re-infarction or non-fatal stroke, and the secondary endpoint of hospitalization for revascularisation by means of a percutaneous coronary intervention (PCI) or coronary artery bypass surgery (CABG). Results: ST-elevation MI was the most frequent presentation among both men and women (85.1 vs. 87.4%, p = ns), but the men had a greater baseline coronary atherosclerotic burden (median Duke Coronary Artery Disease Index: 48 vs. 23; median Syntax score 9 vs. 7; both p < 0.001). The primary composite endpoint occurred less frequently among women (25.7% vs. 37.0%; adjusted hazard ratio: 0.69, 95% CI 0.52-0.91; p = 0.01) despite being less likely to receive treatment with most secondary prevention medications during follow up. Conclusions: There are significant sex-related differences in baseline risk factors and outcomes among patients with early-onset MI: women present with a lower atherosclerotic disease burden and, although they are less frequently prescribed secondary prevention measures, experience better long-term outcomes. Trial Registration: 4272/98 Ospedale Niguarda, Ca' Granda 03/09/1998.

The Role of Epigenetics in Primary Biliary Cholangitis.

Primary Biliary Cholangitis (PBC) is a rare autoimmune disease of the liver, affecting mostly females. There is evidence that epigenetic changes have a pathogenic role in PBC. Epigenetic modifications are related to methylation of CpG DNA islands, post-translational modifications of histone proteins, and non-coding RNAs. In PBC, there are data showing a dysregulation of all these levels, especially in immune cells. In addition, epigenetics seems to be involved in complex phenomena such as X monosomy or abnormalities in the process of X chromosome inactivation, which have been reported in PBC and appear to influence its sex imbalance and pathogenesis. We review here historical data on epigenetic modifications in PBC, present new data, and discuss possible links among X-chromosome abnormalities at a genetic and epigenetic level, PBC pathogenesis, and PBC sex imbalance.

OxDNA to Study Species Interactions.

Molecular ecology uses molecular genetic data to answer traditional ecological questions in biogeography and biodiversity, among others. Several ecological principles, such as the niche hypothesis and the competitive exclusions, are based on the fact that species compete for resources. More in generally, it is now recognized that species interactions play a crucial role in determining the coexistence and abundance of species. However, experimentally controllable platforms, which allow us to study and measure competitions among species, are rare and difficult to implement. In this work, we suggest exploiting a Molecular Dynamics coarse-grained model to study interactions among single strands of DNA, representing individuals of different species, which compete for binding to other oligomers considered as resources. In particular, the well-established knowledge of DNA-DNA interactions at the nanoscale allows us to test the hypothesis that the maximum consecutive overlap between pairs of oligomers measure the species' competitive advantages. However, we suggest that a more complex structure also plays a role in the ability of the species to successfully bind to the target resource oligomer. We complement the simulations with experiments on populations of DNA strands which qualitatively confirm our hypotheses. These tools constitute a promising starting point for further developments concerning the study of controlled, DNA-based, artificial ecosystems.

Needles in Haystacks: Understanding the Success of Selective Pairing of Nucleic Acids.

The effectiveness of several biological and biotechnological processes relies on the remarkably selective pairing of nucleic acids in contexts of molecular complexity. Relevant examples are the on-target binding of primers in genomic PCR and the regulatory efficacy of microRNA via binding on the transcriptome. Here, we propose a statistical framework that enables us to describe and understand such selectivity by means of a model that is extremely cheap from a computational point of view. By re-parametrizing the hybridization thermodynamics on three classes of base pairing errors, we find a convenient way to obtain the free energy of pairwise interactions between nucleic acids. We thus evaluate the hybridization statistics of a given oligonucleotide within a large number of competitive sites that we assume to be random, and we compute the probability of on-target binding. We apply our strategy to PCR amplification and microRNA-based gene regulation, shedding new light on their selectivity. In particular, we show the relevance of the defectless pairing of 3' terminals imposed by the polymerase in PCR selection. We also evaluate the selectivity afforded by the microRNA seed region, thus quantifying the extra contributions given by mechanisms beyond pairing statistics.

Recognition and inhibition of SARS-CoV-2 by humoral innate immunity pattern recognition molecules.

The humoral arm of innate immunity includes diverse molecules with antibody-like functions, some of which serve as disease severity biomarkers in coronavirus disease 2019 (COVID-19). The present study was designed to conduct a systematic investigation of the interaction of human humoral fluid-phase pattern recognition molecules (PRMs) with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Of 12 PRMs tested, the long pentraxin 3 (PTX3) and mannose-binding lectin (MBL) bound the viral nucleocapsid and spike proteins, respectively. MBL bound trimeric spike protein, including that of variants of concern (VoC), in a glycan-dependent manner and inhibited SARS-CoV-2 in three in vitro models. Moreover, after binding to spike protein, MBL activated the lectin pathway of complement activation. Based on retention of glycosylation sites and modeling, MBL was predicted to recognize the Omicron VoC. Genetic polymorphisms at the MBL2 locus were associated with disease severity. These results suggest that selected humoral fluid-phase PRMs can play an important role in resistance to, and pathogenesis of, COVID-19, a finding with translational implications.

Long-term outcomes of early-onset myocardial infarction with non-obstructive coronary artery disease (MINOCA).

BACKGROUND: Acute myocardial infarction with non-obstructive coronary artery disease (MINOCA) is frequent in patients experiencing an early-onset MI, but data concerning its long-term prognosis are limited and conflicting. METHODS: The Italian Genetic Study on Early-onset MI enrolled 2000 patients experiencing a first MI before the age of 45 years, and had a median follow-up of 19.9 years. The composite primary endpoint was cardiovascular (CV) death, non-fatal MI, and non-fatal stroke (MACE); the secondary endpoint was rehospitalisation for coronary revascularisation. RESULTS: MINOCA occurred in 317 patients (15.9%) and, during the follow-up, there was no significant difference in MACE rates between them and the patients with obstructive coronary artery disease (MICAD: 27.8% vs 37.5%; adjusted hazard ratio [HR] 0.79, 95% confidence interval [CI] 0.57-1.09;p = 0.15). The CV death rate was lower in the MINOCA group (4.2% vs 8.4%, HR 0.26, 95%CI 0.08-0.86;p = 0.03), whereas the rates of non-fatal reinfarction (17.3% vs 25.4%; HR 0.76, 95%CI 0.52-1.13;p = 0.18), non-fatal ischemic stroke (9.5% vs 3.7%; HR 1.79, 95%CI 0.87-3.70;p = 0.12), and all-cause mortality (14.1% vs 20.7%, HR 0.73, 95%CI 0.43-1.25;p = 0.26) were not significantly different in the two groups. The rate of rehospitalisation for coronary revascularisation was lower among the MINOCA patients (6.7% vs 27.7%; HR 0.27, 95% CI 0.15-0.47;p < 0.001). CONCLUSIONS: MINOCA is frequent and not benign in patients with early-onset MI. Although there is a lower likelihood of CV death,the long-term risk of MACE and overall mortality is not significantly different from that of MICAD patients.

LINE1 are spliced in non-canonical transcript variants to regulate T cell quiescence and exhaustion.

How gene expression is controlled to preserve human T cell quiescence is poorly understood. Here we show that non-canonical splicing variants containing long interspersed nuclear element 1 (LINE1) enforce naive CD4+ T cell quiescence. LINE1-containing transcripts are derived from CD4+ T cell-specific genes upregulated during T cell activation. In naive CD4+ T cells, LINE1-containing transcripts are regulated by the transcription factor IRF4 and kept at chromatin by nucleolin; these transcripts act in cis, hampering levels of histone 3 (H3) lysine 36 trimethyl (H3K36me3) and stalling gene expression. T cell activation induces LINE1-containing transcript downregulation by the splicing suppressor PTBP1 and promotes expression of the corresponding protein-coding genes by the elongating factor GTF2F1 through mTORC1. Dysfunctional T cells, exhausted in vitro or tumor-infiltrating lymphocytes (TILs), accumulate LINE1-containing transcripts at chromatin. Remarkably, depletion of LINE1-containing transcripts restores TIL effector function. Our study identifies a role for LINE1 elements in maintaining T cell quiescence and suggests that an abundance of LINE1-containing transcripts is critical for T cell effector function and exhaustion.

Impact of chronic exposure to 5-alpha reductase inhibitors on the risk of hospitalization for COVID-19: a case-control study in male population from two COVID-19 regional centers of Lombardy, Italy.

BACKGROUND: There are sex differences in vulnerability to Coronavirus disease 2019 (COVID-19). The coronavirus S protein mediates viral entry into target cells employing the host cellular serine protease TMPRSS2 for S-protein priming. The TMPRSS2 gene expression is responsive to androgen stimulation and it could partially explain sex differences. We hypothesized that men chronically exposed to 5-alpha reductase inhibitors (5ARIs) for benign prostate hyperplasia (BPH) have a lower risk of hospitalization for COVID-19. METHODS: This is a population-based case-control study on consecutive patients positive for SARS-CoV-2 virus who required hospitalization for COVID-19 (cases), age-matched to beneficiaries of the Lombardy Regional Health Service (controls). Data were collected by two high-volume COVID-19 regional centers of Lombardy (Italy). The primary outcome was to compare the prevalence of patients chronically exposed to 5ARIs, who required hospitalization for COVID-19, with the one of controls. RESULTS: Overall, 943 males were enrolled; 45 (4.77%) were exposed to 5ARI. COVID-19 patients aged >55 years under 5ARI treatment were significantly less than expected on the basis of the prevalence of 5ARI treatment among age-matched controls (5.57 vs. 8.14%; P=0.0083, 95% CI: 0.75-3.97%). This disproportion was higher for men aged >65 (7.14 vs. 12.31%; P=0.0001, 95% CI: 2.83-6.97%). Eighteen 5ARIs-patients died; the mean age of men who died was higher than those who did not: 75.98±9.29 vs. 64.78±13.57 (P<0.001). Cox-regression and multivariable models did not show correlation between 5ARIs exposure and protection against intensive care unit admission/death. CONCLUSIONS: Men exposed to 5ARIs might be less vulnerable to severe COVID-19, supporting its use in disease prophylaxis.

DNA Methylation Signature in Monozygotic Twins Discordant for Psoriatic Disease.

Background: Psoriatic disease is a multifactorial inflammatory condition spanning from skin and nail psoriasis (Pso) to spine and joint involvement characterizing psoriatic arthritis (PsA). Monozygotic twins provide a model to investigate genetic, early life environmental exposure and stochastic influences to complex diseases, mainly mediated by epigenetics. Methods: We performed a genome-wide DNA methylation study on whole blood of monozygotic twins from 7 pairs discordant for Pso/PsA using the Infinium Methylation EPIC array (Illumina). MeDiP-qPCR was used to confirm specific signals. Data were replicated in an independent cohort of seven patients with Pso/PsA and 3 healthy controls. Transcriptomic profiling was performed by RNAsequence on the same 7 monozygotic twin pairs. Results: We identified 2,564 differentially methylated positions between psoriatic disease and controls, corresponding to 1,703 genes, 59% within gene bodies. There were 19 regions with at least two DMPs within 1 kb of distance and significant within-pair Δß-values (p < 0.005), among them SNX25, BRG1 and SMAD3 genes, all involved in TGF-ß signaling pathway, were identified. Co-expression analyses on transcriptome data identified IL-6/JAK/STAT3 and TNF-α pathways as important signaling axes involved in the disease, and they also suggested an altered glucose metabolism in patients' immune cells, characteristic of pro-inflammatory T lymphocytes. Conclusion: The study suggests the presence of an epigenetic signature in affected individuals, pointing to genes involved in immunological and inflammatory responses. This result is also supported by transcriptome data, that altogether suggest a higher activation state of the immune system, that could promote the disease status.

MEDTEC Students against Coronavirus: Investigating the Role of Hemostatic Genes in the Predisposition to COVID-19 Severity.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the etiologic agent of the coronavirus disease 2019 (COVID-19) pandemic. Besides virus intrinsic characteristics, the host genetic makeup is predicted to account for the extreme clinical heterogeneity of the disease, which is characterized, among other manifestations, by a derangement of hemostasis associated with thromboembolic events. To date, large-scale studies confirmed that genetic predisposition plays a role in COVID-19 severity, pinpointing several susceptibility genes, often characterized by immunologic functions. With these premises, we performed an association study of common variants in 32 hemostatic genes with COVID-19 severity. We investigated 49,845 single-nucleotide polymorphism in a cohort of 332 Italian severe COVID-19 patients and 1668 controls from the general population. The study was conducted engaging a class of students attending the second year of the MEDTEC school (a six-year program, held in collaboration between Humanitas University and the Politecnico of Milan, allowing students to gain an MD in Medicine and a Bachelor's Degree in Biomedical Engineering). Thanks to their willingness to participate in the fight against the pandemic, we evidenced several suggestive hits (p < 0.001), involving the PROC, MTHFR, MTR, ADAMTS13, and THBS2 genes (top signal in PROC: chr2:127192625:G:A, OR = 2.23, 95%CI = 1.50-3.34, p = 8.77 × 10-5). The top signals in PROC, MTHFR, MTR, ADAMTS13 were instrumental for the construction of a polygenic risk score, whose distribution was significantly different between cases and controls (p = 1.62 × 10-8 for difference in median levels). Finally, a meta-analysis performed using data from the Regeneron database confirmed the contribution of the MTHFR variant chr1:11753033:G:A to the predisposition to severe COVID-19 (pooled OR = 1.21, 95%CI = 1.09-1.33, p = 4.34 × 10-14 in the weighted analysis).

X Chromosome Contribution to the Genetic Architecture of Primary Biliary Cholangitis.

BACKGROUND & AIMS: Genome-wide association studies in primary biliary cholangitis (PBC) have failed to find X chromosome (chrX) variants associated with the disease. Here, we specifically explore the chrX contribution to PBC, a sexually dimorphic complex autoimmune disease. METHODS: We performed a chrX-wide association study, including genotype data from 5 genome-wide association studies (from Italy, United Kingdom, Canada, China, and Japan; 5244 case patients and 11,875 control individuals). RESULTS: Single-marker association analyses found approximately 100 loci displaying P < 5 × 10-4, with the most significant being a signal within the OTUD5 gene (rs3027490; P = 4.80 × 10-6; odds ratio [OR], 1.39; 95% confidence interval [CI], 1.028-1.88; Japanese cohort). Although the transethnic meta-analysis evidenced only a suggestive signal (rs2239452, mapping within the PIM2 gene; OR, 1.17; 95% CI, 1.09-1.26; P = 9.93 × 10-8), the population-specific meta-analysis showed a genome-wide significant locus in East Asian individuals pointing to the same region (rs7059064, mapping within the GRIPAP1 gene; P = 6.2 × 10-9; OR, 1.33; 95% CI, 1.21-1.46). Indeed, rs7059064 tags a unique linkage disequilibrium block including 7 genes: TIMM17B, PQBP1, PIM2, SLC35A2, OTUD5, KCND1, and GRIPAP1, as well as a superenhancer (GH0XJ048933 within OTUD5) targeting all these genes. GH0XJ048933 is also predicted to target FOXP3, the main T-regulatory cell lineage specification factor. Consistently, OTUD5 and FOXP3 RNA levels were up-regulated in PBC case patients (1.75- and 1.64-fold, respectively). CONCLUSIONS: This work represents the first comprehensive study, to our knowledge, of the chrX contribution to the genetics of an autoimmune liver disease and shows a novel PBC-related genome-wide significant locus.

Functional and clinical implications of genetic structure in 1686 Italian exomes.

To reconstruct the phenotypical and clinical implications of the Italian genetic structure, we thoroughly analyzed a whole-exome sequencing data set comprised of 1686 healthy Italian individuals. We found six previously unreported variants with remarkable frequency differences between Northern and Southern Italy in the HERC2, OR52R1, ADH1B, and THBS4 genes. We reported 36 clinically relevant variants (submitted as pathogenic, risk factors, or drug response in ClinVar) with significant frequency differences between Italy and Europe. We then explored putatively pathogenic variants in the Italian exome. On average, our Italian individuals carried 16.6 protein-truncating variants (PTVs), with 2.5% of the population having a PTV in one of the 59 American College of Medical Genetics (ACMG) actionable genes. Lastly, we looked for PTVs that are likely to cause Mendelian diseases. We found four heterozygous PTVs in haploinsufficient genes (KAT6A, PTCH1, and STXBP1) and three homozygous PTVs in genes causing recessive diseases (DPYD, FLG, and PYGM). Comparing frequencies from our data set to other public databases, like gnomAD, we showed the importance of population-specific databases for a more accurate assessment of variant pathogenicity. For this reason, we made aggregated frequencies from our data set publicly available as a tool for both clinicians and researchers (http://nigdb.cineca.it; NIG-ExIT).

Hereditary Hypofibrinogenemia with Hepatic Storage.

Fibrinogen is a 340-kDa plasma glycoprotein constituted by two sets of symmetrical trimers, each formed by the Aα, Bß, and γ chains (respectively coded by the FGA, FGB, and FGG genes). Quantitative fibrinogen deficiencies (hypofibrinogenemia, afibrinogenemia) are rare congenital disorders characterized by low or unmeasurable plasma fibrinogen antigen levels. Their genetic basis is represented by mutations within the fibrinogen genes. To date, only eight mutations, all affecting a small region of the fibrinogen γ chain, have been reported to cause hereditary hypofibrinogenemia with hepatic storage (HHHS), a disorder characterized by protein aggregation in the endoplasmic reticulum, hypofibrinogenemia, and liver disease of variable severity. Here, we will briefly review the clinic characteristics of HHHS patients and the histological feature of their hepatic inclusions, and we will focus on the molecular genetic basis of this peculiar type of coagulopathy.

ACE2 and TMPRSS2 variants and expression as candidates to sex and country differences in COVID-19 severity in Italy.

As the outbreak of coronavirus disease 2019 (COVID-19) progresses, prognostic markers for early identification of high-risk individuals are an urgent medical need. Italy has one of the highest numbers of SARS-CoV-2-related deaths and one of the highest mortality rates. Worldwide, a more severe course of COVID-19 is associated with older age, comorbidities, and male sex. Hence, we searched for possible genetic components of COVID-19 severity among Italians by looking at expression levels and variants in ACE2 and TMPRSS2 genes, crucial for viral infection.Exome and SNP-array data from a large Italian cohort were used to compare the rare-variants burden and polymorphisms frequency with Europeans and East Asians. Moreover, we looked into gene expression databases to check for sex-unbalanced expression.While we found no significant evidence that ACE2 is associated with disease severity/sex bias, TMPRSS2 levels and genetic variants proved to be possible candidate disease modulators, prompting for rapid experimental validations on large patient cohorts.