Analisi di metilazione gene specifica in pazienti affetti da malattia di Alzheimer

La malattia di Alzheimer (MA) è una malattia neurodegenerativa che rappresenta da sola circa i due terzi di tutte le forme di demenza. La MA è caratterizzata clinicamente da un progressivo deterioramento delle funzioni cognitive e da atrofia cerebrale che si localizza prevalentemente a livello della neocorteccia. A livello microscopico, il cervello di un individuo affetto è caratterizzato dalla presenza extracellulare di placche senili, costituite da un eccessivo deposito del peptide β-amiloide, che deriva dal taglio proteolitico della proteina precursore dell’amiloide (APP), ad opera degli enzimi β-secretasi (BACE1) e γ-secretasi. Inoltre si riscontra la presenza di grovigli neurofibrillari intracellulari, dovuti all’iperfosforilazione della proteina tau. Circa l’1-5% dei casi della malattia è riconducibile a mutazioni causative a carico dei geni PSEN1, PSEN2 (codificanti per componenti della γ-secretasi) e APP (codificante la proteina APP), mentre nella maggior parte dei casi si manifesta in maniera sporadica, verosimilmente in seguito all’interazione di fattori di suscettibilità genetici, il più noto dei quali è l’APOε4, e fattori di origine ambientale, come lo stile di vita, l’alimentazione e l’esposizione ad inquinanti. In particolare sempre più studi hanno mostrato come un deficit di acido folico con compromissione del ciclo dei folati, fondamentale per la sintesi di importanti composti cellulari e per la metilazione di DNA e altre macromolecole, sia associato ad un aumentato rischio di sviluppare la MA. I fattori ambientali promuoverebbero infiammazione e stress ossidativo in grado di indurre danno al DNA e compromissione dei meccanismi per la sua riparazione. Sempre più studi hanno inoltre evidenziato che diversi fattori ambientali sono in grado di indurre alterazioni epigenetiche in grado di contribuire allo sviluppo della MA; in particolare, alterazioni della metilazione del DNA sono state osservate sia a livello di tessuto cerebrale post-mortem che in cellule di sangue periferico di individui con MA. Nel presente lavoro di tesi sono stati ricercati possibili marcatori epigenetici periferici in soggetti affetti da MA. A tale scopo sono stati condotti due studi. In un primo studio (studio 1) sono stati valutati i livelli di metilazione di geni coinvolti nel processamento del peptide β-amiloide (PSEN1 e BACE1), nella metilazione del DNA (DNMT1, DNMT3A e DNMT3B) e nel metabolismo dei folati (MTHFR) mediante la tecnica Methylation-Sensitive High Resolution Melting (MS-HRM) e i valori ottenuti sono stati correlati con fattori di rischio per la MA, come l’età, il sesso, biomarcatori del ciclo dei folati, quali l’omocisteina, folati e vitamina B12 e presenza del genotipo APOE ε4. Inoltre in un sottogruppo di questi individui sono state analizzate le forze di connessione tra le variabili analizzate mediante l’applicazione di reti neurali artificiali (ANN). Dato che è ben noto che nel cervello e nei tessuti periferici di individui con MA vi è sia un aumentato danno al DNA che una alterazione dei meccanismi della sua riparazione, in un secondo studio (studio 2) sono stati valutati i livelli di metilazione di 25 geni che codificano per proteine coinvolte nella riparazione del DNA, mediante la MS-HRM e mediante un array di metilazione. Tutti i geni analizzati nello studio 1 sono risultati essere ipometilati, con un livello di metilazione inferiore al 5%, tranne il gene MTHFR che ha mostrato dei livelli di metilazione compresi tra il 5 e il 75%. Nessuna differenza significativa nei livelli di metilazione dei geni analizzati è stata osservata tra i pazienti e i controlli. Inoltre, l’elaborazione dei risultati mediante l’applicazione delle ANN ha evidenziato interessanti correlazioni tra i livelli di metilazione dei geni PSEN1, BACE1, MTHFR e delle DNMT e i livelli plasmatici di omocisteina, folati e vitamina B12. Nello studio 2 sia la MS-HRM che l’array di metilazione hanno mostrato che tutti i geni coinvolti nella riparazione del DNA analizzati sono altamente ipometilati sia nei pazienti con MA che nei controlli, e non è stata evidenziata nessuna differenza tra i due gruppi in esame. In conclusione, dal momento che non sono state osservate differenze tra i pazienti e i controlli, l’analisi di metilazione dei geni investigati non sembrerebbe essere un utile biomarker nella discriminazione tra pazienti e controlli. Tuttavia, nel loro insieme i risultati ottenuti suggeriscono che c’è una stretta correlazione tra i livelli di metilazione dei geni analizzati a livello del sangue periferico e i livelli plasmatici di omocisteina, folati e vitamina B12, noti biomarcatori associati alla MA

Mitoepigenetics and Neurodegenerative Diseases

Mitochondrial impairment and increased oxidative stress are common features in neurodegenerative disorders, leading researchers to speculate that epigenetic changes in the mitochondrial DNA (mitoepigenetics) could contribute to neurodegeneration. The few studies performed so far to address this issue revealed impaired methylation levels of the mitochondrial regulatory region (D-loop region) in both animal models, postmortem brain regions, or circulating blood cells of patients with Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis. Those studies also revealed that mtDNA D-loop methylation levels are subjected to a dynamic regulation within the progression of the neurodegenerative process, could be affected by certain neurodegenerative disease-causative mutations, and are inversely correlated with the mtDNA copy number. The methylation levels of other mtDNA regions than the D-loop have been scarcely investigated in human specimens from patients with neurodegenerative disorders or in animal models of the disease, and evidence of impaired methylation levels is often limited to a single study, making it difficult to clarify their correlation with mitochondrial dynamics and gene expression levels in these disorders. Overall, the preliminary results of the studies performed so far are encouraging making mitoepigenetics a timely and attractive field of investigation, but additional research is warranted to clarify the connections among epigenetic changes occurring in the mitochondrial genome, mitochondrial DNA dynamics and gene expression, and the neurodegenerative process

Genotoxic and epigenetic mechanisms in arsenic carcinogenicity

Arsenic is a human carcinogen with weak mutagenic properties that induces tumors through mechanisms not yet completely understood. People worldwide are exposed to arsenic-contaminated drinking water, and epidemiological studies showed a high percentage of lung, bladder, liver, and kidney cancer in these populations. Several mechanisms by which arsenical compounds induce tumorigenesis were proposed including genotoxic damage and chromosomal abnormalities. Over the past decade, a growing body of evidence indicated that epigenetic modifications have a role in arsenic-inducing adverse effects on human health. The main epigenetic mechanisms are DNA methylation in gene promoter regions that regulate gene expression, histone tail modifications that regulate the accessibility of transcriptional machinery to genes, and microRNA activity (noncoding RNA able to modulate mRNA translation). The "double capacity" of arsenic to induce mutations and epimutations could be the main cause of arsenic-induced carcinogenesis. The aim of this review is to better clarify the mechanisms of the initiation and/or the promotion of arsenic-induced carcinogenesis in order to understand the best way to perform an early diagnosis and a prompt prevention that is the key point for protecting arsenic-exposed population. Studies on arsenic-exposed population should be designed in order to examine more comprehensively the presence and consequences of these genetic/epigenetic alterations

Artificial Neural Networks Link One-Carbon Metabolism to Gene-Promoter Methylation in Alzheimer's Disease

Background: There is increasing interest in DNA methylation studies in Alzheimer's disease (AD), but little is still known concerning the relationship between gene-promoter methylation and circulating biomarkers of one-carbon metabolism in patients. Objective: To detect the connections among circulating folate, homocysteine (hcy) and vitamin B12 levels and promoter methylation levels of PSEN1, BACE1, DNMT1, DNMT3A, DNMT3B, and MTHFR genes in blood DNA. Methods: We applied a data mining system called Auto Contractive Map to an existing database of 100 AD and 100 control individuals. Results: Low vitamin B12 was linked to the AD condition, to low folates, and to high hcy. Low PSEN1 methylation was linked to low folate levels as well as to low promoter methylation of BACE1 and DNMTs genes. Low hcy was linked to controls, to high folates and vitamin B12, as well as to high methylation levels of most of the studied genes. Conclusions: The present pilot study suggests that promoter methylation levels of the studied genes are linked to circulating levels of folates, hcy, and vitamin B12

Association study between the DNMT3A -448A>G polymorphism and risk of Alzheimer's disease in Caucasians of Italian origin

Increasing evidence points to an epigenetic contribution in Alzheimer's disease (AD) pathogenesis. In this regard, variants and polymorphisms of DNA methyltransferase genes (DNMTs) are being investigated for their contribution to cognitive decline and dementia, but results are still scarce or controversial. In the present study we genotyped 710 Caucasian subjects of Italian descent, including 320 late-onset AD (LOAD) patients, 70 individuals with amnestic Mild Cognitive Impairment (MCI), and 320 matched healthy controls, for the presence of a functional DNMT3A -448A>G (rs1550117) polymorphism, searching for association with disease risk. In addition, we searched for correlation between the studied polymorphism and circulating levels of folate, homocysteine (hcy) and vitamin B12, all involved in DNA methylation reactions and available from 189 LOAD patients and 186 matched controls. Both allele and genotype frequencies of rs1550117 were closely similar between MCI, LOAD and control subjects, and no association with dementia or pre-dementia conditions was observed. Plasma hcy levels were significantly higher (p = 0.04) and serum folate levels significantly lower (p = 0.01) in LOAD than in controls, but no difference in circulating folate, hcy or vitamin B12 levels was seen between carriers and non-carriers of the minor DNMT3A -448A allele. Collectively, present results confirmed previous associations of increased hcy and decreased folate with LOAD risk, but do not support an association between the DNMT3A -448A>G polymorphism and AD in our population

Increase in Mitochondrial D-Loop Region Methylation Levels in Mild Cognitive Impairment Individuals

Methylation levels of the mitochondrial displacement loop (D-loop) region have been reported to be altered in the brain and blood of Alzheimer's disease (AD) patients. Moreover, a dynamic D-loop methylation pattern was observed in the brain of transgenic AD mice along with disease progression. However, investigations on the blood cells of AD patients in the prodromal phases of the disease have not been performed so far. The aim of this study was to analyze D-loop methylation levels by means of the MS-HRM technique in the peripheral blood cells of 14 mild cognitive impairment (MCI) patients, 18 early stage AD patients, 70 advanced stage AD patients, and 105 healthy control subjects. We found higher D-loop methylation levels in MCI patients than in control subjects and AD patients. Moreover, higher D-loop methylation levels were observed in control subjects than in AD patients in advanced stages of the disease, but not in those at early stages. The present pilot study shows that peripheral D-loop methylation levels differ in patients at different stages of AD pathology, suggesting that further studies deserve to be performed in order to validate the usefulness of D-loop methylation analysis as a peripheral biomarker for the early detection of AD

Complessi pincer NCN di Au(III) quali possibili agenti antitumorali

I composti di oro sono da molto tempo utilizzati in medicina (es. nella cura dell’artrite reumatoide). Attualmente rivestono grande importanza come possibili agenti anticancro, settore dominato dai complessi di platino(II): composti di oro(III), isoelettronici ed isostrutturali con i complessi di platino(II), dopo un esordio abbastanza deludente, hanno ridestato l’interesse come antitumorali a partire dalla metà degli anni ’90 ed ora la letteratura è ricca di lavori che ne riportano l’attività citostatica sia in vitro che in vivo. Da tempo ci occupiamo di complessi pincer tipo [M(N^C^N)Cl] M=Pd(II), Pt(II)3 e da poco abbiamo esteso questa nostra ricerca anche all’Au(III): malgrado il largo interesse per la chimica dei pincer complessi NCN i derivati di oro sono molto rari

Prenatal Environmental Stressors and DNA Methylation Levels in Placenta and Peripheral Tissues of Mothers and Neonates Evaluated by Applying Artificial Neural Networks

Exposure to environmental stressors during pregnancy plays an important role in influencing subsequent susceptibility to certain chronic diseases through the modulation of epigenetic mechanisms, including DNA methylation. Our aim was to explore the connections between environmental exposures during gestation with DNA methylation of placental cells, maternal and neonatal buccal cells by applying artificial neural networks (ANNs). A total of 28 mother-infant pairs were enrolled. Data on gestational exposure to adverse environmental factors and on mother health status were collected through the administration of a questionnaire. DNA methylation analyses at both gene-specific and global level were analyzed in placentas, maternal and neonatal buccal cells. In the placenta, the concentrations of various metals and dioxins were also analyzed. Analysis of ANNs revealed that suboptimal birth weight is associated with placental H19 methylation, maternal stress during pregnancy with methylation levels of NR3C1 and BDNF in placentas and mother's buccal DNA, respectively, and exposure to air pollutants with maternal MGMT methylation. Associations were also observed between placental concentrations of lead, chromium, cadmium and mercury with methylation levels of OXTR in placentas, HSD11B2 in maternal buccal cells and placentas, MECP2 in neonatal buccal cells, and MTHFR in maternal buccal cells. Furthermore, dioxin concentrations were associated with placental RELN, neonatal HSD11B2 and maternal H19 gene methylation levels. Current results suggest that exposure of pregnant women to environmental stressors during pregnancy could induce aberrant methylation levels in genes linked to several pathways important for embryogenesis in both the placenta, potentially affecting foetal development, and in the peripheral tissues of mothers and infants, potentially providing peripheral biomarkers of environmental exposure

Comparison Study of MS-HRM and Pyrosequencing Techniques for Quantification of APC and CDKN2A Gene Methylation

There is increasing interest in the development of cost-effective techniques for the quantification of DNA methylation biomarkers. We analyzed 90 samples of surgically resected colorectal cancer tissues for APC and CDKN2A promoter methylation using methylation sensitive-high resolution melting (MS-HRM) and pyrosequencing. MS-HRM is a less expensive technique compared with pyrosequencing but is usually more limited because it gives a range of methylation estimates rather than a single value. Here, we developed a method for deriving single estimates, rather than a range, of methylation using MS-HRM and compared the values obtained in this way with those obtained using the gold standard quantitative method of pyrosequencing. We derived an interpolation curve using standards of known methylated/ unmethylated ratio (0%, 12.5%, 25%, 50%, 75%, and 100% of methylation) to obtain the best estimate of the extent of methylation for each of our samples. We observed similar profiles of methylation and a high correlation coefficient between the two techniques. Overall, our new approach allows MS-HRM to be used as a quantitative assay which provides results which are comparable with those obtained by pyrosequencing