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.

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.

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.

Mycobacterium tuberculosis Drives Expansion of Low-Density Neutrophils Equipped With Regulatory Activities.

In human tuberculosis (TB) neutrophils represent the most commonly infected phagocyte but their role in protection and pathology is highly contradictory. Moreover, a subset of low-density neutrophils (LDNs) has been identified in TB, but their functions remain unclear. Here, we have analyzed total neutrophils and their low-density and normal-density (NDNs) subsets in patients with active TB disease, in terms of frequency, phenotype, functional features, and gene expression signature. Full-blood counts from Healthy Donors (H.D.), Latent TB infected, active TB, and cured TB patients were performed. Frequency, phenotype, burst activity, and suppressor T cell activity of the two different subsets were assessed by flow cytometry while NETosis and phagocytosis were evaluated by confocal microscopy. Expression analysis was performed by using the semi-quantitative RT-PCR array technology. Elevated numbers of total neutrophils and a high neutrophil/lymphocyte ratio distinguished patients with active TB from all the other groups. PBMCs of patients with active TB disease contained elevated percentages of LDNs compared with those of H.D., with an increased expression of CD66b, CD33, CD15, and CD16 compared to NDNs. Transcriptomic analysis of LDNs and NDNs purified from the peripheral blood of TB patients identified 12 genes differentially expressed: CCL5, CCR5, CD4, IL10, LYZ, and STAT4 were upregulated, while CXCL8, IFNAR1, NFKB1A, STAT1, TICAM1, and TNF were downregulated in LDNs, as compared to NDNs. Differently than NDNs, LDNs failed to phagocyte live Mycobacterium tuberculosis (M. tuberculosis) bacilli, to make oxidative burst and NETosis, but caused significant suppression of antigen-specific and polyclonal T cell proliferation which was partially mediated by IL-10. These insights add a little dowel of knowledge in understanding the pathogenesis of human TB.

Gene-gene interactions among coding genes of iron-homeostasis proteins and APOE-alleles in cognitive impairment diseases.

Cognitive impairments of different aetiology share alterations in iron and lipid homeostasis with mutual relationships. Since iron and cholesterol accumulation impact on neurodegenerative disease, the associated gene variants are appealing candidate targets for risk and disease progression assessment. In this light, we explored the role of common single nucleotide polymorphisms (SNPs) in the main iron homeostasis genes and in the main lipoprotein transporter gene (APOE) in a cohort of 765 patients with dementia of different origin: Alzheimer's disease (AD) n = 276; vascular dementia (VaD), n = 255; mild cognitive impairment (MCI), n = 234; and in normal controls (n = 1086). In details, four genes of iron homeostasis (Hemochromatosis (HFE: C282Y, H63D), Ferroportin (FPN1: -8CG), Hepcidin (HAMP: -582AG), Transferrin (TF: P570S)), and the three major alleles of APOE (APOE2, APOE3, APOE4) were analyzed to explore causative interactions and synergies. In single analysis, HFE 282Y allele yielded a 3-fold risk reduction in the whole cohort of patients (P<0.0001), confirmed in AD and VaD, reaching a 5-fold risk reduction in MCI (P = 0.0019). The other iron SNPs slightly associated with risk reduction whereas APOE4 allele resulted in increased risk, reaching more than 7-fold increased risk in AD homozygotes (P = 0.001), confirmed to a lower extent in VaD and MCI (P = 0.038 and P = 0.013 respectively) as well as in the whole group (P<0.0001). Comparisons of Mini Mental State Examination (MMSE) among AD showed appreciable lowering in APOE4 carriers (P = 0.038), confirmed in the whole cohort of patients (P = 0.018). In interaction analysis, the HFE 282Y allele completely extinguished the APOE4 allele associated risk. Conversely, the coexistence in patients of a substantial number of iron SNPs accrued the APOE4 detrimental effect on MMSE. Overall, the analysis highlighted how a specific iron-allele burden, defined as different combinations of iron gene variants, might have different effects on cognitive impairment and might modulate the effects of established genetic risk factors such as APOE4. Our results suggest that established genetic risk factors might be affected by specific genetic backgrounds, making patients differently suited to manage iron accumulation adding new genetic insights in neurodegeneration. The recently recognized interconnections between iron and lipids, suggest that these pathways might share more than expected. We therefore extended to additional iron gene variants the newly proposed influencing mechanisms that HFE gene has on cholesterol metabolism. Our results have a strong translational potential promoting new pharmacogenetics studies on therapeutic target identification aimed at optimally tuning brain iron levels.

Fibrinogen as a Pleiotropic Protein Causing Human Diseases: The Mutational Burden of Aα, Bß, and γ Chains.

Fibrinogen is a highly pleiotropic protein that is involved in the final step of the coagulation cascade, wound healing, inflammation, and angiogenesis. Heterozygous mutations in Aα, Bß, or γ fibrinogen-chain genes (FGA, FGB, FGG) have been described as being responsible for fibrinogen deficiencies (hypofibrinogenemia, hypo-dysfibrinogenemia, dysfibrinogenemia) and for more rare conditions, such as fibrinogen storage disease and hereditary renal amyloidosis. Instead, biallelic mutations have been associated with afibrinogenemia/severe hypofibrinogenemia, i.e., the severest forms of fibrinogen deficiency, affecting approximately 1-2 cases per million people. However, the "true" prevalence for these conditions on a global scale is currently not available. Here, we defined the mutational burden of the FGA, FGB, and FGG genes, and estimated the prevalence of inherited fibrinogen disorders through a systematic analysis of exome/genome data from ~140,000 individuals belonging to the genome Aggregation Database. Our analysis showed that the world-wide prevalence for recessively-inherited fibrinogen deficiencies could be 10-fold higher than that reported so far (prevalence rates vary from 1 in 106 in East Asians to 24.5 in 106 in non-Finnish Europeans). The global prevalence for autosomal-dominant fibrinogen disorders was estimated to be ~11 in 1000 individuals, with heterozygous carriers present at a frequency varying from 3 every 1000 individuals in Finns, to 1-2 every 100 individuals among non-Finnish Europeans and Africans/African Americans. Our analysis also allowed for the identification of recurrent (i.e., FGG-p.Ala108Gly, FGG-Thr47Ile) or ethnic-specific mutations (e.g., FGB-p.Gly103Arg in Admixed Americans, FGG-p.Ser245Phe in Africans/African Americans).

The Characterization of GSDMB Splicing and Backsplicing Profiles Identifies Novel Isoforms and a Circular RNA That Are Dysregulated in Multiple Sclerosis.

Abnormalities in alternative splicing (AS) are emerging as recurrent features in autoimmune diseases (AIDs). In particular, a growing body of evidence suggests the existence of a pathogenic association between a generalized defect in splicing regulatory genes and multiple sclerosis (MS). Moreover, several studies have documented an unbalance in alternatively-spliced isoforms in MS patients possibly contributing to the disease etiology. In this work, using a combination of PCR-based techniques (reverse-transcription (RT)-PCR, fluorescent-competitive, real-time, and digital RT-PCR assays), we investigated the alternatively-spliced gene encoding Gasdermin B, GSDMB, which was repeatedly associated with susceptibility to asthma and AIDs. The in-depth characterization of GSDMB AS and backsplicing profiles led us to the identification of an exonic circular RNA (ecircRNA) as well as of novel GSDMB in-frame and out-of-frame isoforms. The non-productive splicing variants were shown to be downregulated by the nonsense-mediated mRNA decay (NMD) in human cell lines, suggesting that GSDMB levels are significantly modulated by NMD. Importantly, both AS isoforms and the identified ecircRNA were significantly dysregulated in peripheral blood mononuclear cells of relapsing-remitting MS patients compared to controls, further supporting the notion that aberrant RNA metabolism is a characteristic feature of the disease.

miR-634 is a Pol III-dependent intronic microRNA regulating alternative-polyadenylated isoforms of its host gene PRKCA.

BACKGROUND: The protein kinase C alpha (PRKCA) gene, coding for a Th17-cell-selective kinase, shows a complex splicing pattern, with at least 2 stable alternative transcripts characterized by an alternative upstream polyadenylation site. Polymorphisms in this gene were associated with several conditions, including multiple sclerosis, asthma, schizophrenia, and cancer. The presence of a microRNA (miRNA), i.e. miR-634, within intron 15 of the PRKCA gene, suggests the intriguing possibility that this miRNA might play a role in the susceptibility to these pathologies. METHODS: Here, we characterized miR-634 expression profile and searched for its putative targets using a combination of RT-PCR and gene reporter assays. RESULTS: The quantitative analysis of PRKCA and miR-634 transcripts in a panel of human tissues and cell lines revealed discordant expression profiles, suggesting the presence of an independent miR-634 promoter and/or a possible direct role of miR-634 in modulating PRKCA expression. Functional studies demonstrated the existence of a miRNA-specific promoter, which was shown to be Pol-III-dependent. Furthermore, transfection experiments showed that miR-634 is able to target its host gene by specifically down-regulating the shorter alternative-polyadenylated isoforms. CONCLUSIONS: MiR-634 is a Pol III-dependent intronic miRNA, which could target its host gene through a "first-order" negative feedback. GENERAL SIGNIFICANCE: MiR-634 is one of the few characterized examples of Pol-III-dependent intronic miRNAs. Its independent transcription from the host gene suggests caution in using expression profiles of host genes as proxies for the expression of the corresponding intronic miRNAs.

Notch1 regulates chemotaxis and proliferation by controlling the CC-chemokine receptors 5 and 9 in T cell acute lymphoblastic leukaemia.

Tumour cells often express deregulated profiles of chemokine receptors that regulate cancer cell migration and proliferation. Notch1 pathway activation is seen in T cell acute lymphoblastic leukaemia (T-ALL) due to the high frequency of Notch1 mutations affecting approximately 60% of patients, causing ligand-independent signalling and/or prolonging Notch1 half-life. We have investigated the possible regulative role of Notch1 on the expression and function of chemokine receptors CCR5, CCR9 and CXCR4 that play a role in determining blast malignant properties and localization of extramedullary infiltrations in leukaemia. We inhibited the pathway through γ-Secretase inhibitor and Notch1 RNA interference and analysed the effect on the expression and function of chemokine receptors. Our results indicate that γ-Secretase inhibitor negatively regulates the transcription level of the CC chemokine receptors 5 and 9 in T-ALL cell lines and patients' primary leukaemia cells, leaving CXCR4 expression unaltered. The Notch pathway also controls CCR5- and CCR9-mediated biological effects, ie chemotaxis and proliferation. Furthermore, engaging CCR9 through CCL25 administration rescues proliferation inhibition associated with abrogation of Notch activity. Finally, through RNA interference we demonstrated that the oncogenic isoform in T-ALL, Notch1, plays a role in controlling CCR5 and CCR9 expression and functions. These findings suggest that Notch1, acting in concert with chemokine receptors pathways, may provide leukaemia cells with proliferative advantage and specific chemotactic abilities, therefore influencing tumour cell progression and localization.

Genetic association and altered gene expression of mir-155 in multiple sclerosis patients.

Multiple sclerosis (MS) is a complex autoimmune disease of the central nervous system characterized by chronic inflammation, demyelination, and axonal damage. As microRNA (miRNA)-dependent alterations in gene expression in hematopoietic cells are critical for mounting an appropriate immune response, miRNA deregulation may result in defects in immune tolerance. In this frame, we sought to explore the possible involvement of miRNAs in MS pathogenesis by monitoring the differential expression of 22 immunity-related miRNAs in peripheral blood mononuclear cells of MS patients and healthy controls, by using a microbead-based technology. Three miRNAs resulted >2 folds up-regulated in MS vs controls, whereas none resulted down-regulated. Interestingly, the most up-regulated miRNA (mir-155; fold change = 3.30; P = 0.013) was previously reported to be up-regulated also in MS brain lesions. Mir-155 up-regulation was confirmed by qPCR experiments. The role of mir-155 in MS susceptibility was also investigated by genotyping four single nucleotide polymorphisms (SNPs) mapping in the mir-155 genomic region. A haplotype of three SNPs, corresponding to a 12-kb region encompassing the last exon of BIC (the B-cell Integration Cluster non-coding RNA, from which mir-155 is processed), resulted associated with the disease status (P = 0.035; OR = 1.36, 95% CI = 1.05-1.77), suggesting that this locus strongly deserves further investigations.