Studio di associazione tra i polimorfismi DNMT3B -149 C>T, DNMT3B -579 G>T e la Miastenia Gravis.

RIASSUNTO La Miastenia Gravis (MG) è una malattia neuromuscolare di tipo autoimmunitario in cui si ha un difetto nella trasmissione dell’impulso nervoso a livello della sinapsi tra i motoneuroni colinergici e il muscolo scheletrico. La MG colpisce la muscolatura volontaria, in particolare quella del cranio e degli arti, causandone la perdita di tono e forza. Tale patologia può insorgere a qualsiasi età e interessa entrambi i sessi, anche se, come per tutte le patologie autoimmuni, le donne sono maggiormente affette. Esistono varie forme di MG, ma quella principalmente analizzata in questo studio è detta Miastenia autoimmune acquisita. In questo caso, alcuni anticorpi autoreattivi, diretti principalmente contro il recettore acetilcolinico, vanno ad interferire con la trasmissione sinaptica a livello della giunzione neuromuscolare. Il risultato è una trasmissione parziale dell’impulso nervoso con conseguente limitazione della possibilità di contrazione muscolare e una rapida diminuzione della forza. Caratteristica peculiare di tale patologia è il fatto che i pazienti mostrano ognuno una diversa sintomatologia, la cui gravità può variare anche nel corso della singola giornata. Come per altre malattie complesse, l’ambiente potrebbe giocare un ruolo nell’insorgenza della MG tramite modificazioni epigenetiche, quali ad esempio la metilazione del DNA. Nell’uomo la metilazione è affidata alle DNA-metiltransferasi (DNMT), enzimi che possono effettuare una metilazione di mantenimento o una metilazione de-novo. Oggetto di questo lavoro di tesi è la DNMT3B, dato il suo coinvolgimento nella metilazione de-novo del genoma; è stato così effettuato uno studio caso-controllo per valutare il possibile contributo di due polimorfismi, DNMT3B -149C>T e DNMT3B -579G>T, nel rischio di MG. La tecnica usata per l’analisi dei polimorfismi è la PCR-RFLP, che consiste nell’amplificazione del frammento di nostro interesse e nella successiva digestione enzimatica che consente la discriminazione dei diversi genotipi attraverso una corsa elettroforetica e successiva visualizzazione ai raggi UV. Dai risultati ottenuti è stato osservato che soggetti eterozigoti per il polimorfismo DNMT3B -149C>T mostrano una significativa diminuzione del rischio di sviluppare Miastenia Gravis, mentre non è stata osservata alcuna associazione statisticamente significativa per il polimorfismo DNMT3B -579G>T. Effettuando una suddivisione in base al sesso dei soggetti analizzati, è emersa una differenza statisticamente significativa tra casi e controlli nel gruppo delle donne e non nei soggetti di sesso maschile; in particolare il genotipo eterozigote -149CT risulta associato a rischio di sviluppare MG solo nei soggetti di sesso femminile. I presenti risultati suggeriscono un possibile contributo del polimorfismo DNMT3B -149C>T alla MG, in particolare nelle donne

Studio e narrazione del paesaggio montano della Lunigiana: due progetti per la Val di Vara (SP)

Il contributo presenta due progetti realizzati in Val di Vara (SP) con approccio multidisciplinare e diacronico, coinvolgendo amministrazioni e popolazione locali: “TraMonti” (2011-2012) e “Sesta Godano” (2015- 2016). La metodologia applicata ha previsto lo studio di fonti scritte, cartografiche e toponomastiche, seguito dal censimento delle emergenze materiali e dalla raccolta di fonti orali. L’importazione di tali dati su GIS ha permesso di acquisire un quadro della storia di un’area per più motivi complessa da indagare. “TraMonti” ha incluso una fase di narrazione dei risultati, comprensiva di un webGIS consultabile a scopo di ricerca, valorizzazione e gestione del territorio; il lavoro su Sesta Godano è in fase di edizione dei dati raccolti, dei quali si presenta una prima sintesi

MicroRNAs in Hypertrophic, Arrhythmogenic and Dilated Cardiomyopathy

MicroRNAs (miRNAs) are a class of non-coding RNAs of about 20 nucleotides in length, involved in the regulation of many biochemical pathways in the human body. The level of miRNAs in tissues and circulation can be deregulated because of altered pathophysiological mechanisms; thus, they can be employed as biomarkers for different pathological conditions, such as cardiac diseases. This review summarizes published findings of these molecular biomarkers in the three most common structural cardiomyopathies: human dilated, arrhythmogenic and hypertrophic cardiomyopathy

Post-Mortem Cardiac Magnetic Resonance for the Diagnosis of Hypertrophic Cardiomyopathy

Background: Post-mortem cardiac magnetic resonance (PMCMR) is an emerging tool supporting forensic medicine for the identification of the causes of cardiac death, such as hypertrophic cardiomyopathy (HCM). We proposed a new method of PMCMR to diagnose HCM despite myocardial rigor mortis. Methods: We performed CMR in 49 HCM patients, 30 non-HCM hypertrophy, and 32 healthy controls. In cine images, rigor mortis was simulated by the analysis of the cardiac phase corresponding to 25% of diastole. Left ventricular mass, mean, and standard deviation (SD) of WT, maximal WT, minimal WT, and their difference were compared for the identification of HCM. These parameters were validated at PMCMR, evaluating eight hearts with HCM, 10 with coronary artery disease, and 10 with non-cardiac death. Results: The SD of WT with a cut-off of > 2.4 had the highest accuracy to identify HCM (AUC 0.95, 95% CI = 0.89-0.98). This was particularly evident in the female population of HCM (AUC=0.998), with 100% specificity (95% CI = 85-100%) and 96% sensitivity (95% CI = 79-99%). Using this parameter, at PMCMR, all of the eight patients with HCM were correctly identified with no false positives. Conclusions: PMCMR allows identification of HCM as the cause of sudden death using the SD of WT > 2.4 as the diagnostic parameter

Unveiling a sudden unexplained death case by whole exome sequencing and bioinformatic analysis

Abstract Background Sudden unexplained death (SUD) refers to cases of sudden death where autopsy fails to identify any cardiac or extracardiac underlying cause. Guideline‐directed standard genetic testing identifies a disease‐causing mutation in less than one‐third of cases of SUD. Conversely, whole exome sequencing (WES) may provide the key to solve most cases of SUD even after several years from the subject's death. Methods We report on a case of sudden unexpected death of a 37‐year‐old male, with inconclusive autopsy conducted 14 years ago. A recent reevaluation through WES was performed on DNA extracted from left ventricular samples. A multiple step process including several “in silico” tools was applied to identify potentially pathogenic variants. Data analysis was based on a 562 gene panel, including 234 candidate genes associated with sudden cardiac death or heart diseases, with the addition of 328 genes highly expressed in the heart. WebGestalt algorithms were used for association enrichment analysis of all genes with detected putative pathogenic variants. Results WES analysis identified four potentially pathogenic variants: RYR2:c.12168G>T, TTN:c.11821C>T (rs397517804), MYBPC3:c.1255C>T (rs368770848), and ACADVL:c.848T>C (rs113994167). WebGestalt algorithms indicated that their combination holds an unfavorable arrhythmic susceptibility which conceivably caused the occurrence of the events leading to our subject's sudden death. Conclusion Associating WES technique with online prediction algorithms may allow the recognition of genetic mutations potentially responsible for otherwise unexplained deaths

Expression of MicroRNAs in Sepsis-Related Organ Dysfunction: A Systematic Review

Sepsis is a critical condition characterized by increased levels of pro-inflammatory cytokines and proliferating cells such as neutrophils and macrophages in response to microbial pathogens. Such processes lead to an abnormal inflammatory response and multi-organ failure. MicroRNAs (miRNA) are single-stranded non-coding RNAs with the function of gene regulation. This means that miRNAs are involved in multiple intracellular pathways and thus contribute to or inhibit inflammation. As a result, their variable expression in different tissues and organs may play a key role in regulating the pathophysiological events of sepsis. Thanks to this property, miRNAs may serve as potential diagnostic and prognostic biomarkers in such life-threatening events. In this narrative review, we collect the results of recent studies on the expression of miRNAs in heart, blood, lung, liver, brain, and kidney during sepsis and the molecular processes in which they are involved. In reviewing the literature, we find at least 122 miRNAs and signaling pathways involved in sepsis-related organ dysfunction. This may help clinicians to detect, prevent, and treat sepsis-related organ failures early, although further studies are needed to deepen the knowledge of their potential contribution

Post-mortem CMR in a model of sudden death due to myocardial ischemia: validation with connexin-43

Objectives: We sought to evaluate the effectiveness of post-mortem cardiac magnetic resonance (PM-CMR) for the identification of myocardial ischemia as cause of sudden cardiac death (SCD) when the time interval between the onset of ischemia and SCD is ≤ 90 min. Methods: PM-CMR was performed in 8 hearts explanted from pigs with spontaneous death caused by occlusion of the left anterior descending coronary artery: 4 with SCD after ≤ 40 min of coronary occlusion and 4 between 40 and 90 min. PM-CMR included conventional T1 and T2-weighted image and T1, T2, and T2* mapping techniques. Imaging data were compared and validated with immunohistochemical evaluation of the altered proportion and redistribution of phosphorylated versus non-phosphorylated connexin 43 (CX43 and npCX43, respectively), an established molecular marker of myocardial ischemia. Results: At T2-weighted images, the ischemic core was hypointense (core/remote ratio 0.67 ± 0.11) and surrounded by and hyperintense border zone. Compared to remote myocardium, the ischemic core had higher T1 (p = 0.0008), and lower T2 (p = 0.007) and T2* (p = 0.002). Cytoplasmatic npX43 and the npCX43/CX43 ratio were significantly higher in animals deceased > 40 min than in others. Conclusion: PM-CMR can reliably detect early signs of myocardial damage induced by ischemia, based on conventional pulse sequences complemented by a novel ad hoc application of quantitative mapping techniques. Key points: • Post-mortem MRI may help to understand cause of sudden cardiac death. • Post-mortem MRI allows detection of signs of myocardial ischemia as cause of sudden cardiac death within 90 and 40 min following coronary occlusion as demonstrated in a pig model of myocardial ischemia. • Signs of myocardial ischemia using conventional and mapping MRI technique are associated with the immunohistochemical changes of phosphorylated and dephosphorylated connexin-43 which is an established molecular marker of myocardial ischemia