BNT162b2 Booster Dose Elicits a Robust Antibody Response in Subjects with Abdominal Obesity and Previous SARS-CoV-2 Infection.

Little is known about the long-term durability of the induced immune response in subjects with obesity, particularly in those with an abdominal distribution of adipose tissue. We evaluated SARS-CoV-2-specific antibody responses after BNT162b2 vaccine booster dose, comparing individuals with and without abdominal obesity (AO), discerning between individuals previously infected or not. IgG-TrimericS were measured in 511 subjects at baseline, on the 21st day after vaccine dose 1, and at 1, 3, 6, and 9 months from dose 2, and at 1 and 3 months following the booster dose. To detect SARS-CoV-2 infection, nucleocapsid antibodies were measured at baseline and at the end of the study. Multivariable linear regression evaluated the three-month difference in the absolute variation in IgG-TrimericS levels from booster dose, showing AO and SARS-CoV-2 infection status interactions (p = 0.016). Regardless of possible confounding factors and IgG-TrimericS levels at the booster dose, AO is associated with a higher absolute change in IgG-TrimericS in prior infected individuals (p = 0.0125). In the same regression model, no interaction is highlighted using BMI (p = 0.418). The robust response in the development of antibodies after booster dose, observed in people with AO and previous infection, may support the recommendations to administer a booster dose in this population group.

Rationale and design of the CV-PREVITAL study: an Italian multiple cohort randomised controlled trial investigating innovative digital strategies in primary cardiovascular prevention.

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.

Reducing contrast agent residuals in hospital wastewater: the GREENWATER study protocol.

The potential enviromental impact of iodinated (ICAs) and gadolinium-based contrast agents (GBCAs) have recently come under scrutiny, considering the current nonselective wastewater treatment. However, their rapid excretion after intravenous administration could allow their potential recovery by targeting hospital sewage. The GREENWATER study aims to appraise the effective quantities of ICAs and GBCAs retrievable from patients' urine collected after computed tomography (CT) and magnetic resonance imaging (MRI) exams, selecting ICA/GBCA per-patient urinary excretion and patients' acceptance rate as study endpoints. Within a prospective, observational, single-centre, 1-year framework, we will enrol outpatients aged ≥ 18 years, scheduled to perform contrast-enhanced CT or MRI, willing to collect post-examination urine in dedicated canisters by prolonging their hospital stay to 1 h after injection. Collected urine will be processed and partially stored in the institutional biobank. Patient-based analysis will be performed for the first 100 CT and 100 MRI patients, and then, all analyses will be conducted on the pooled urinary sample. Quantification of urinary iodine and gadolinium will be performed with spectroscopy after oxidative digestion. The evaluation of the acceptance rate will assess the "environmental awareness" of patients and will aid to model how procedures to reduce ICA/GBCA enviromental impact could be adapted in different settings. Key points • Enviromental impact of iodinated and gadolinium-based contrast agents represents a growing point of attention.• Current wastewater treatment is unable to retrieve and recycle contrast agents.• Prolonging hospital stay may allow contrast agents retrieval from patients' urine.• The GREENWATER study will assess the effectively retrievable contrast agents' quantities.• The enrolment acceptance rate will allow to evaluate patients' "green sensitivity".

Impact of BNT162b2 Booster Dose on SARS-CoV-2 Anti-Trimeric Spike Antibody Dynamics in a Large Cohort of Italian Health Care Workers.

Accurate studies on the dynamics of Pfizer-Biontech BNT162b2-induced antibodies are crucial to better tailor booster dose administration depending on age, comorbidities, and previous natural infection with SARS-CoV-2. To date, little is known about the durability and kinetics of antibody titers months after receiving a booster dose. In this work, we studied the dynamic of anti-Trimeric Spike (anti-TrimericS) IgG titer in the healthcare worker population of a large academic hospital in Northern Italy, in those who had received two vaccine doses plus a booster dose. Blood samples were collected on the day of dose 1, dose 2, then 1 month, 3 months, and 6 months after dose 2, the day of the administration of the booster dose, then 1 month and 3 months after the booster dose. The vaccination immunogenicity was evaluated by dosing anti-TrimericS IgG titer, which was further studied in relation to SARS-CoV-2 infection status, age, and sex. Our results suggest that after the booster dose, the anti-TrimericS IgG production was higher in the subjects that were infected only after the completion of the vaccination cycle, compared to those that were infected both before and after the vaccination campaign. Moreover, the booster dose administration exerts a leveling effect, mitigating the differences in the immunogenicity dependent on sex and age.

Association of miR-144 levels in the peripheral blood with COVID-19 severity and mortality.

Coronavirus disease-2019 (COVID-19) can be asymptomatic or lead to a wide symptom spectrum, including multi-organ damage and death. Here, we explored the potential of microRNAs in delineating patient condition and predicting clinical outcome. Plasma microRNA profiling of hospitalized COVID-19 patients showed that miR-144-3p was dynamically regulated in response to COVID-19. Thus, we further investigated the biomarker potential of miR-144-3p measured at admission in 179 COVID-19 patients and 29 healthy controls recruited in three centers. In hospitalized patients, circulating miR-144-3p levels discriminated between non-critical and critical illness (AUCmiR-144-3p = 0.71; p = 0.0006), acting also as mortality predictor (AUCmiR-144-3p = 0.67; p = 0.004). In non-hospitalized patients, plasma miR-144-3p levels discriminated mild from moderate disease (AUCmiR-144-3p = 0.67; p = 0.03). Uncontrolled release of pro-inflammatory cytokines can lead to clinical deterioration. Thus, we explored the added value of a miR-144/cytokine combined analysis in the assessment of hospitalized COVID-19 patients. A miR-144-3p/Epidermal Growth Factor (EGF) combined score discriminated between non-critical and critical hospitalized patients (AUCmiR-144-3p/EGF = 0.81; p < 0.0001); moreover, a miR-144-3p/Interleukin-10 (IL-10) score discriminated survivors from nonsurvivors (AUCmiR-144-3p/IL-10 = 0.83; p < 0.0001). In conclusion, circulating miR-144-3p, possibly in combination with IL-10 or EGF, emerges as a noninvasive tool for early risk-based stratification and mortality prediction in COVID-19.

Cell environment shapes TDP-43 function with implications in neuronal and muscle disease.

TDP-43 (TAR DNA-binding protein 43) aggregation and redistribution are recognised as a hallmark of amyotrophic lateral sclerosis and frontotemporal dementia. As TDP-43 inclusions have recently been described in the muscle of inclusion body myositis patients, this highlights the need to understand the role of TDP-43 beyond the central nervous system. Using RNA-seq, we directly compare TDP-43-mediated RNA processing in muscle (C2C12) and neuronal (NSC34) mouse cells. TDP-43 displays a cell-type-characteristic behaviour targeting unique transcripts in each cell-type, which is due to characteristic expression of RNA-binding proteins, that influence TDP-43's performance and define cell-type specific splicing. Among splicing events commonly dysregulated in both cell lines, we identify some that are TDP-43-dependent also in human cells. Inclusion levels of these alternative exons are altered in tissues of patients suffering from FTLD and IBM. We therefore propose that TDP-43 dysfunction contributes to disease development either in a common or a tissue-specific manner.

Antibody responses to BNT162b2 mRNA vaccine: Infection-naïve individuals with abdominal obesity warrant attention.

OBJECTIVE: The excess of visceral adipose tissue might hinder and delay immune response. How people with abdominal obesity (AO) will respond to mRNA vaccines against SARS-CoV-2 is yet to be established. SARS-CoV-2-specific antibody responses were evaluated after the first and second dose of the BNT162b2 mRNA vaccine, comparing the response of individuals with AO with the response of those without, and discerning between individuals with or without prior infection. METHODS: Immunoglobulin G (IgG)-neutralizing antibodies against the Trimeric complex (IgG-TrimericS) were measured at four time points: at baseline, at day 21 after vaccine dose 1, and at 1 and 3 months after dose 2. Nucleocapsid antibodies were assessed to detect prior SARS-CoV-2 infection. Waist circumference was measured to determine AO. RESULTS: Between the first and third month after vaccine dose 2, the drop in IgG-TrimericS levels was more remarkable in individuals with AO compared with those without AO (2.44-fold [95% CI: 2.22-2.63] vs. 1.82-fold [95% CI: 1.69-1.92], respectively, p < 0.001). Multivariable linear regression confirmed this result after inclusion of assessed confounders (p < 0.001). CONCLUSIONS: The waning antibody levels in individuals with AO may further support recent recommendations to offer booster vaccines to adults with high-risk medical conditions, including obesity, and particularly to those with a more prevalent AO phenotype.

Viral Agents and Systemic Levels of Inflammatory Cytokines in Vulnerable and Stable Atherosclerotic Carotid Plaques.

BACKGROUND: To investigate the presence of genetic material of viral agents and the serum level of inflammatory cytokines in patients submitted to carotid endarterectomy having vulnerable versus stable atherosclerotic plaques. METHODS: Data of patients consecutively submitted to carotid endarterectomy for a significant stenosis from July 2019 to December 2019 were prospectively collected. The genetic material of Epstein-Barr (EBV), CitoMegalo (CMV), Herpes Simplex (HSV), Varicella-Zoster (VZV) and Influenza (IV) Viruses was searched in the patient's plaques, both in the "mid" of the plaque and in an adjacent lateral portion of no-plaque area. The serum levels of TNF-α, IL-1ß, IL-6, IL10 and CCL5 were determined. The obtained results were then correlated to the histologic vulnerability of the removed carotid plaque. P values < 0.05 were considered statistically significant. RESULTS: Data of 50 patients were analyzed. A vulnerable plaque was found in 31 patients (62%). The genome of CMV, HSV, VZV and IV was not found in any of the vascular samples, while the EBV genome was found in the "mid" of 2 vulnerable plaques, but not in their respective control area. Eighty-two percent of patients who did not receive anti-IV vaccination (23/28) had vulnerable carotid plaque, compared with 36% of vaccinated patients (8/22, P = 0.001). Serum levels of TNF-α and IL-6 were higher in patients with a vulnerable plaque compared to patients with a stable plaque (73.6 ± 238.2 vs. 3.9 ± 13.1 pg/ml, P= 0.01, and 45.9 ± 103.6 vs. 10.1 ± 25.3 pg/ml, P= 0.01, respectively), independent of comorbidities, viral exposure or flu vaccination. CONCLUSIONS: The EBV genome was found in the "core" of 2 vulnerable carotid plaques, but not in their respective adjacent control. Influenza vaccination was associated with a lower incidence of carotid plaque vulnerability. Serum levels of TNF-α and IL-6 were higher in patients with a vulnerable plaque compared to patients with a stable plaque.

First Family of MATR3-Related Distal Myopathy From Italy: The Role of Muscle Biopsy in the Diagnosis and Characterization of a Still Poorly Understood Disease.

Mutations in the MATR3 gene are associated to distal myopathy with vocal cord and pharyngeal weakness (VCPDM), as well as familiar and sporadic motor neuron disease. To date, 12 VCPDM families from the United States, Germany, Japan, Bulgary, and France have been described in the literature. Here we report an Italian family with a propositus of a 40-year-old woman presenting progressive bilateral foot drop, rhinolalia, and distal muscular atrophy, without clinical signs of motor neuron affection. Her father, deceased some years before, presented a similar distal myopathy phenotype, while her 20-year-old son is asymptomatic. Myopathic changes with vacuolization were observed in muscle biopsy from the propositus. These results, together with the peculiar clinical picture, lead to MATR3 gene sequencing, which revealed a heterozygous p.S85C mutation in the propositus. The same mutation was found in her son. Over a 5-year follow-up, progression is mild in the propositus, while her son remains asymptomatic. Clinical, radiological, and pathological data of our propositus are presented and compared to previously reported cases of VCPDM. VCPDM turns out to be a quite homogenous phenotype of late-onset myopathy associated to p.S85C mutation in MATR3 gene. MATR3-related pathology, encompassing myopathy and motor neuron disease, represents an illustrative example of multisystem proteinopathy (MSP), such as other diseases associated to mutations in VCP, HNRNPA2B1, HNRNPA1, and SQSTM1 genes. The present report contributes to a further characterization of this still poorly understood pathology and points out the diagnostic utility of muscle biopsy in challenging cases.

Co-occurrence of DMPK expansion and CLCN1 mutation in a patient with myotonia.

INTRODUCTION: Myotonic disorders are a group of diseases affecting the muscle, in different ways. Myotonic dystrophy type 1 (DM1) is related to (CTG)n expansion in the 3-untranslated region of the dystrophia myotonica protein kinase (DMPK) gene and is the most frequent and disabling form, causing muscular, visibility, respiratory, and cardiac impairment. Non-dystrophic myotonias (NDMs) affect the skeletal muscle alone. In particular, mutations in the chloride channel (CLCN1) gene cause myotonia congenita (MC), which can have autosomal dominant or recessive inheritance. CASE REPORT: We describe a patient with a family history of asymptomatic or paucisymptomatic myotonia, who presented handgrip myotonia which sharply reduced after mexiletine administration. Molecular analysis showed both a paternally inherited DMPK expansion and a maternally inherited CLCN1 mutation. CONCLUSIONS: Only one other similar case was reported so far; however, the segregation of the two mutations and the characteristics of the muscle were not studied. Since our patient lacked the classical phenotypical and muscle histopathological characteristics of DM1 and showed mild splicing alterations despite a pathogenic DMPK expansion and the nuclear accumulation of toxic RNA, we may speculate that the co-occurrence of a CLCN1 mutation could have attenuated the severity of DM1 phenotype.

A 14-Year Italian Experience in DM2 Genetic Testing: Frequency and Distribution of Normal and Premutated CNBP Alleles.

Myotonic dystrophy type 2 (DM2) is a multisystemic disorder caused by a (CCTG) n in intron 1 of the CNBP gene. The CCTG repeat tract is part of a complex (TG) v (TCTG) w (CCTG) x (NCTG) y (CCTG) z motif generally interrupted in CNBP healthy range alleles. Here we report our 14-year experience of DM2 postnatal genetic testing in a total of 570 individuals. The DM2 locus has been analyzed by a combination of SR-PCR, TP-PCR, LR-PCR, and Sanger sequencing of CNBP alleles. DM2 molecular diagnosis has been confirmed in 187/570 samples analyzed (32.8%) and is mainly associated with the presence of myotonia in patients. This set of CNBP alleles showed unimodal distribution with 25 different alleles ranging from 108 to 168 bp, in accordance with previous studies on European populations. The most frequent CNBP alleles consisted of 138, 134, 140, and 136 bps with an overall locus heterozygosity of 90%. Sequencing of 103 unexpanded CNBP alleles in DM2-positive patients revealed that (CCTG)5(NCTG)3(CCTG)7 and (CCTG)6(NCTG)3(CCTG)7 are the most common interruption motifs. We also characterized five CNBP premutated alleles with (CCTG) n repetitions from n = 36 to n = 53. However, the molecular and clinical consequences in our cohort of samples are not unequivocal. Data that emerged from this study are representative of the Italian population and are useful tools for National and European centers offering DM2 genetic testing and counseling.

Soluble Receptor for Advanced Glycation End Products and Its Forms in COVID-19 Patients with and without Diabetes Mellitus: A Pilot Study on Their Role as Disease Biomarkers.

The receptor for advanced glycation end products (RAGE), a well-known player of diabetes mellitus (DM)-related morbidities, was supposed to be involved in coronavirus disease-19 (COVID-19), but no data exist about COVID-19, DM, and the soluble RAGE (sRAGE) forms. We quantified total sRAGE and its forms, the endogenously secretory esRAGE and the membrane-cleaved cRAGE, in COVID-19 patients with and without DM and in healthy individuals to explore how COVID-19 may affect these molecules and their potential role as biomarkers. Circulating sRAGE and esRAGE were quantified by enzyme-linked-immunosorbent assays. cRAGE was obtained by subtracting esRAGE from total sRAGE. sRAGE, esRAGE, cRAGE, and the cRAGE/esRAGE ratio did not differ between DM and non-DM patients and had the same trend when compared to healthy individuals. Levels of total sRAGE, cRAGE, and cRAGE/esRAGE ratio were upregulated, while esRAGE was downregulated. The lack of difference between DM and non-DM COVID-19 patients in the levels of sRAGE and its forms supports the hypothesis that in COVID-19 the RAGE system is modulated regardless of glycemic control. Identifying how sRAGE and its forms associate to COVID-19 prognosis and the potential of RAGE as a therapeutic target to control inflammatory burden seem of relevance to help treatment of COVID-19.

Covid-19-Associated Coagulopathy: Biomarkers of Thrombin Generation and Fibrinolysis Leading the Outcome.

Background: Coronavirus Disease 2019 (COVID-19)-associated coagulopathy is characterized by a prothrombotic state not yet comprehensively studied. We investigated the coagulation pattern of patients with COVID-19 acute respiratory distress syndrome (ARDS), comparing patients who survived to those who did not. Methods: In this prospective cohort study on 20 COVID-19 ARDS patients, the following biomarkers were measured: thrombin generation (prothrombin fragment 1 + 2 (PF 1 + 2)), fibrinolysis activation (tissue plasminogen activator (tPA)) and inhibition (plasminogen activator inhibitor 2 (PAI-2)), fibrin synthesis (fibrinopeptide A) and fibrinolysis magnitude (plasmin-antiplasmin complex (PAP) and D-dimers). Measurements were done upon intensive care unit (ICU) admission and after 10-14 days. Results: There was increased thrombin generation; modest or null release of t-PA; and increased levels of PAI-2, fibrinopeptide A, PAP and D-dimers. At baseline, nonsurvivors had a significantly (p = 0.014) higher PAI-2/PAP ratio than survivors (109, interquartile range (IQR) 18.1-216, vs. 8.7, IQR 2.9-12.6). At follow-up, thrombin generation was significantly (p = 0.025) reduced in survivors (PF 1 + 2 from 396 pg/mL, IQR 185-585 to 237 pg/mL, IQR 120-393), whereas it increased in nonsurvivors. Fibrinolysis inhibition at follow-up remained stable in survivors and increased in nonsurvivors, leading to a significant (p = 0.026) difference in PAI-2 levels (161 pg/mL, IQR 50-334, vs. 1088 pg/mL, IQR 177-1565). Conclusion: Severe patterns of COVID-19 ARDS are characterized by a thrombin burst and the consequent coagulation activation. Mechanisms of fibrinolysis regulation appear unbalanced toward fibrinolysis inhibition. This pattern ameliorates in survivors, whereas it worsens in nonsurvivors.

Rare Disease: Cardiac Risk Assessment With MRI in Patients With Myotonic Dystrophy Type 1.

Introduction: To evaluate myocardial strain and extracellular volume in myotonic dystrophy type 1 (DM1) patients as potential imaging biomarkers of subclinical cardiac pathology. Materials and methods: We retrospectively analyzed 9 DM1 patients without apparent cardiac disease who had undergone cardiac magnetic resonance at our center. Patients were age- and sex-matched with healthy controls. The Mann-Whitney U test was used to compare cardiac strain between the two groups. The t-test was used to compare the extracellular volume obtained in DM1 patients with that in healthy subject. Spearman's ρ was used for studying the associations among imaging parameters. Results: Global cardiac strain (median -19.1%; IQR -20.5%, -16.5%) in DM1 patients was lower (p = 0.011) than that in controls (median-21.7%; IQR-22.7%,-21.3%). Global extracellular volume in DM1 patients (median 32.3%; IQR 29.3%,36.8%) was significantly (p = 0.008) higher than that reported in literature in healthy subjects (median 25.6%; IQR 19.9%,31.9%). Global cardiac strain showed a strong, positive correlation with septal strain (ρ = 0.767, p = 0.016) and with both global (ρ = 0.733 p = 0.025) and septal extracellular volume (ρ = 0.767, p = 0.016). Discussion: The increase in cardiac extracellular volume and decrease in strain are signs of early cardiac pathology in DM1. Physicians dealing with DM1 may take into consideration cardiac magnetic resonance as a screening tool to identify early cardiac involvement in this condition.

TNNT2 Missplicing in Skeletal Muscle as a Cardiac Biomarker in Myotonic Dystrophy Type 1 but Not in Myotonic Dystrophy Type 2.

Cardiac involvement is one of the most important manifestations of the multisystemic phenotype of patients affected by myotonic dystrophy (DM) and represents the second cause of premature death. Molecular mechanisms responsible for DM cardiac defects are still unclear; however, missplicing of the cardiac isoform of troponin T (TNNT2) and of the cardiac sodium channel (SCN5A) genes might contribute to the reduced myocardial function and conduction abnormalities seen in DM patients. Since, in DM skeletal muscle, the TNNT2 gene shows the same aberrant splicing pattern observed in cardiac muscle, the principal aim of this work was to verify if the TNNT2 aberrant fetal isoform expression could be secondary to myopathic changes or could reflect the DM cardiac phenotype. Analysis of alternative splicing of TNNT2 and of several genes involved in DM pathology has been performed on muscle biopsies from patients affected by DM type 1 (DM1) or type 2 (DM2) with or without cardiac involvement. Our analysis shows that missplicing of muscle-specific genes is higher in DM1 and DM2 than in regenerating control muscles, indicating that these missplicing could be effectively important in DM skeletal muscle pathology. When considering the TNNT2 gene, missplicing appears to be more evident in DM1 than in DM2 muscles since, in DM2, the TNNT2 fetal isoform appears to be less expressed than the adult isoform. This evidence does not seem to be related to less severe muscle histopathological alterations that appear to be similar in DM1 and DM2 muscles. These results seem to indicate that the more severe TNNT2 missplicing observed in DM1 could not be related only to myopathic changes but could reflect the more severe general phenotype compared to DM2, including cardiac problems that appear to be more severe and frequent in DM1 than in DM2 patients. Moreover, TNNT2 missplicing significantly correlates with the QRS cardiac parameter in DM1 but not in DM2 patients, indicating that this splicing event has good potential to function as a biomarker of DM1 severity and it should be considered in pharmacological clinical trials to monitor the possible effects of different therapeutic approaches on skeletal muscle tissues.

Aberrant regulation of epigenetic modifiers contributes to the pathogenesis in patients with selenoprotein N-related myopathies.

Congenital myopathies are early onset, slowly progressive neuromuscular disorders of variable severity. They are genetically and phenotypically heterogeneous and caused by pathogenic variants in several genes. Multi-minicore Disease, one of the more common congenital myopathies, is frequently caused by recessive variants in either SELENON, encoding the endoplasmic reticulum glycoprotein selenoprotein N or RYR1, encoding a protein involved in calcium homeostasis and excitation-contraction coupling. The mechanism by which recessive SELENON variants cause Multiminicore disease (MmD) is unclear. Here, we extensively investigated muscle physiological, biochemical and epigenetic modifications, including DNA methylation, histone modification, and noncoding RNA expression, to understand the pathomechanism of MmD. We identified biochemical changes that are common in patients harboring recessive RYR1 and SELENON variants, including depletion of transcripts encoding proteins involved in skeletal muscle calcium homeostasis, increased levels of Class II histone deacetylases (HDACs) and DNA methyltransferases. CpG methylation analysis of genomic DNA of patients with RYR1 and SELENON variants identified >3,500 common aberrantly methylated genes, many of which are involved in calcium signaling. These results provide the proof of concept for the potential use of drugs targeting HDACs and DNA methyltransferases to treat patients with specific forms of congenital myopathies.

Dysregulation of Circular RNAs in Myotonic Dystrophy Type 1.

Circular RNAs (circRNAs) constitute a recently re-discovered class of non-coding RNAs functioning as sponges for miRNAs and proteins, affecting RNA splicing and regulating transcription. CircRNAs are generated by "back-splicing", which is the linking covalently of 3'- and 5'-ends of exons. Thus, circRNA levels might be deregulated in conditions associated with altered RNA-splicing. Significantly, growing evidence indicates their role in human diseases. Specifically, myotonic dystrophy type 1 (DM1) is a multisystemic disorder caused by expanded CTG repeats in the DMPK gene which results in abnormal mRNA-splicing. In this investigation, circRNAs expressed in DM1 skeletal muscles were identified by analyzing RNA-sequencing data-sets followed by qPCR validation. In muscle biopsies, out of nine tested, four transcripts showed an increased circular fraction: CDYL, HIPK3, RTN4_03, and ZNF609. Their circular fraction values correlated with skeletal muscle strength and with splicing biomarkers of disease severity, and displayed higher values in more severely affected patients. Moreover, Receiver-Operating-Characteristics curves of these four circRNAs discriminated DM1 patients from controls. The identified circRNAs were also detectable in peripheral-blood-mononuclear-cells (PBMCs) and the plasma of DM1 patients, but they were not regulated significantly. Finally, increased circular fractions of RTN4_03 and ZNF609 were also observed in differentiated myogenic cell lines derived from DM1 patients. In conclusion, this pilot study identified circRNA dysregulation in DM1 patients.

Aberrant insulin receptor expression is associated with insulin resistance and skeletal muscle atrophy in myotonic dystrophies.

Myotonic dystrophy type 1 (DM1) and type 2 (DM2) are autosomal dominant multisystemic disorders linked to two different genetic loci and characterized by several features including myotonia, muscle atrophy and insulin resistance. The aberrant alternative splicing of insulin receptor (IR) gene and post-receptor signalling abnormalities have been associated with insulin resistance, however the precise molecular defects that cause metabolic dysfunctions are still unknown. Thus, the aims of this study were to investigate in DM skeletal muscle biopsies if beyond INSR missplicing, altered IR protein expression could play a role in insulin resistance and to verify if the lack of insulin pathway activation could contribute to skeletal muscle wasting. Our analysis showed that DM skeletal muscle exhibits a lower expression of the insulin receptor in type 1 fibers which can contribute to the defective activation of the insulin pathway. Moreover, the aberrant insulin signalling activation leads to a lower activation of mTOR and to an increase in MuRF1 and Atrogin-1/MAFbx expression, possible explaining DM skeletal muscle fiber atrophy. Taken together our data indicate that the defective insulin signalling activation can contribute to skeletal muscle features in DM patients and are probably linked to an aberrant specific-fiber type expression of the insulin receptor.

Neuropsychological and Psychological Functioning Aspects in Myotonic Dystrophy Type 1 Patients in Italy.

Introduction: Myotonic Dystrophy Type 1 (DM1) is an autosomal dominant genetic illness, characterized by a progressive loss of strength. Important deficits in cognitive functioning and a significant prevalence of psychiatric disorders have been previously reported. Methods:A neuropsychological and psychological assessment was carried out in 31 DM1 patients (61% males) in order to measure the cognitive functioning and explore their personality profiles. The MMSE Mini-Mental State Examination, Frontal Assessment Battery (FAB), ENB-2 Battery assessing memory (short term, long term and working memory), integration capacities, visual-spatial ability, attention (selective, divided, shifting/switching) executive functions, praxis, discrimination and logic capabilities and psychopathology Symptom Check List 90-R (SCL-90-R) were administered. The neuropsychological and psychological evaluation of DM1 patients was carried out taking into consideration the clinical parameters (CTG repeat, age at onset, disease duration, Muscular Impairment Rate Scale (MIRS), Medical Research Council Scale (MRC) and the Epworth Sleepiness Scales (EPS)). Results: Regarding psychopathology 19.4% of patients scored a moderate or high level of symptoms intensity index (GSI), 12.9% reported a high number of symptoms (PST) and 16.1% reported a high intensity level of the perceived symptoms (PSDI). Fatigue and daytime sleepiness resulted as being associated with higher levels of psychoticism (PSY). Only 1 patient reported a severe impairment in the spatial and temporal orientation, memory, language, praxis, attention and calculation. Longer disease duration was also associated with cognitive impairment evaluated through ENB-2 (p < 0.05). Discussions and Conclusions:There are indications of the utility of neuropsychological and psychological screening and support for these patients and their families due to the link between disease duration and cognitive performances. A proposal of a clinical protocol, with an illustration of a clinical case report of a family is presented.

SCN4A as modifier gene in patients with myotonic dystrophy type 2.

A patient with an early severe myotonia diagnosed for Myotonic Dystrophy type 2 (DM2) was found bearing the combined effects of DM2 mutation and Nav1.4 S906T substitution. To investigate the mechanism underlying his atypical phenotype,whole-cell patch-clamp in voltage- and current-clamp mode was performed in myoblasts and myotubes obtained from his muscle biopsy. Results characterizing the properties of the sodium current and of the action potentials have been compared to those obtained in muscle cells derived from his mother, also affected by DM2, but without the S906T polymorphism. A faster inactivation kinetics and a +5 mV shift in the availability curve were found in the sodium current recorded in patient's myoblasts compared to his mother. 27% of his myotubes displayed spontaneous activity. Patient's myotubes showing a stable resting membrane potential had a lower rheobase current respect to the mother's while the overshoot and the maximum slope of the depolarizing phase of action potential were higher. These findings suggest that SCN4A polymorphisms may be responsible for a higher excitability of DM2 patients sarcolemma, supporting the severe myotonic phenotype observed. We suggest SCN4A as a modifier factor and that its screening should be performed in DM2 patients with uncommon clinical features.