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The presence of 5-hydroxymethyl cytosine in DNA has been previously associated with ageing. Using in silico analysis of normal liver samples we presently observed that in 5-hydroxymethyl cytosine sequences, DNA methylation is dependent on the co-presence of G-quadruplexes and palindromes. This association exhibits discrete patterns depending on G-quadruplex and palindrome densities. DNase-Seq data show that 5-hydroxymethyl cytosine sequences are common among liver nucleosomes (p < 2.2x10-16) and threefold more frequent than nucleosome sequences. Nucleosomes lacking palindromes and potential G-quadruplexes are rare in vivo (1%) and nucleosome occupancy potential decreases with increasing G-quadruplexes. Palindrome distribution is similar to that previously reported in nucleosomes. In low and mixed complexity sequences 5-hydroxymethyl cytosine is frequently located next to three elements: G-quadruplexes or imperfect G-quadruplexes with CpGs, or unstable hairpin loops (TCCCAY6TGGGA) mostly located in antisense strands or finally A-/T-rich segments near these motifs. The high frequencies and selective distribution of pentamer sequences (including TCCCA, TGGGA) probably indicate the positive contribution of 5-hydroxymethyl cytosine to stabilize the formation of structures unstable in the absence of this cytosine modification. Common motifs identified in all total 5-hydroxymethyl cytosine-containing sequences exhibit high homology to recognition sites of several transcription factor families: homeobox, factors involved in growth, mortality/ageing, cancer, neuronal function, vision, and reproduction. We conclude that cytosine hydroxymethylation could play a role in the recognition of sequences with G-quadruplexes/palindromes by forming epigenetically regulated DNA 'springs' and governing expansions or compressions recognized by different transcription factors or stabilizing nucleosomes. The balance of these epigenetic elements is lost in hepatocellular carcinoma.
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Quadruplex G , Neoplasias , 5-Metilcitosina/análogos & derivados , Envelhecimento , Metilação de DNA , Humanos , Fatores de TranscriçãoRESUMO
The extended and diverse interference of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in multiple host functions and the diverse associated symptoms implicate its involvement in fundamental cellular regulatory processes. The activity of ten-eleven translocase 2 (TET2) responsible for selective DNA demethylation, has been recently identified as a regulator of endogenous virus inactivation and viral invasion, possibly by proteasomal deregulation of the TET2/TET3 activities. In a recent report, we presented a detailed list of factors that can be affected by TET activity, including recognition of zinc finger protein binding sites and bimodal promoters, by enhancing the flexibility of adjacent sequences. In this review, we summarize the TET-associated processes and factors that could account for SARS-CoV-2 diverse symptoms. Moreover, we provide a correlation for the observed virus-induced symptoms that have been previously associated with TET activities by in vitro and in vitro studies. These include early hypoxia, neuronal regulation, smell and taste development, liver, intestinal, and cardiomyocyte differentiation. Finally, we propose that the high mortality of SARS-CoV-2 among adult patients, the different clinical symptoms of adults compared to children, the higher risk of patients with metabolic deregulation, and the low mortality rates among women can all be accounted for by the complex balance of the three enzymes with TET activity, which is developmentally regulated. This activity is age-dependent, related to telomere homeostasis and integrity, and associated with X chromosome inactivation via (de)regulation of the responsible XIST gene expression.
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BACKGROUND: Type 1 diabetes (T1D) has been associated with a higher fracture risk due to alterations in bone structure and metabolism. On the other hand, the important role of the RANKL/OPG/RANK signaling axis in bone physiology is well established. The aim of this study was to evaluate the levels of receptor activator of nuclear factor kappa-B ligand (RANKL), receptor activator of nuclear factor kappa-B (RANK) and plasma osteoprotegerin (OPG) levels, in T1D youngsters and to investigate factors that could influence the OPG/RANK/RANKL signaling axis such as 25-hydroxy vitamin D [25(OH) D], parathormone (PTH) and age. METHODS: Serum RANKL, RANK, 25(OH) D, PTH levels and plasma OPG levels, were measured in 71 youngsters with T1D and 50 healthy controls matched for age and gender. RESULTS: Plasma OPG levels were significantly lower (p = 0.025) in T1D patients compared to controls. Serum RANKL levels were significantly higher (p = 0.037), while no differences were observed in serum RANK levels (p = 0.946) between the two groups. Serum 25(OH) D levels found significantly decreased (p < 0.001) while serum PTH levels were significantly elevated (p < 0.001) in T1D patients than in controls. CONCLUSIONS: Our results demonstrated that OPG and RANKL may be promising biomarkers for T1D patients. However, their circulating levels were associated with several factors including PTH, 25(OH) D and therefore, may represent an integrative biomarker for a variety of endocrine signaling disturbances observed in T1D.
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Diabetes Mellitus Tipo 1/sangue , Osteoprotegerina/sangue , Hormônio Paratireóideo/sangue , Ligante RANK/sangue , Receptor Ativador de Fator Nuclear kappa-B/sangue , Vitamina D/sangue , Adolescente , Biomarcadores/sangue , Estudos de Casos e Controles , Criança , Pré-Escolar , Diabetes Mellitus Tipo 1/diagnóstico , Progressão da Doença , Feminino , Humanos , Masculino , Prognóstico , Valores de ReferênciaRESUMO
DNA methylation studies have been reformed with the advent of single-base resolution arrays and bisulfite sequencing methods, enabling deeper investigation of methylation-mediated mechanisms. In addition to these advancements, numerous bioinformatics tools address important computational challenges, covering DNA methylation calling up to multi-modal interpretative analyses. However, contrary to the analytical frameworks that detect driver mutational signatures, the identification of putatively actionable epigenetic events remains an unmet need. The present work describes a novel computational framework, called MeinteR, that prioritizes critical DNA methylation events based on the following hypothesis: critical aberrations of DNA methylation more likely occur on a genomic substrate that is enriched in cis-acting regulatory elements with distinct structural characteristics, rather than in genomic "deserts". In this context, the framework incorporates functional cis-elements, e.g. transcription factor binding sites, tentative splice sites, as well as conformational features, such as G-quadruplexes and palindromes, to identify critical epigenetic aberrations with potential implications on transcriptional regulation. The evaluation on multiple, public cancer datasets revealed significant associations between the highest-ranking loci with gene expression and known driver genes, enabling for the first time the computational identification of high impact epigenetic changes based on high-throughput DNA methylation data.
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Metilação de DNA/genética , Conformação de Ácido Nucleico , Sequências Reguladoras de Ácido Nucleico/genética , Software , Animais , Neoplasias da Mama/genética , Carcinoma Hepatocelular/genética , Bases de Dados Genéticas , Epigênese Genética , Feminino , Quadruplex G , Regulação Neoplásica da Expressão Gênica , Genoma Humano , Estudo de Associação Genômica Ampla , Humanos , Neoplasias Hepáticas/genética , Camundongos , Mutação/genética , Ratos , Fluxo de TrabalhoRESUMO
Horvath's epigenetic clock consists of 353 CpGs whose methylation levels can accurately predict the age of individuals. Using bioinformatics analysis, we investigated the conformation, energy characteristics and presence of tentative splice sites of the sequences surrounding the epigenetic clock CpGs, in relation to the median methylation changes in different ages, the presence of CpG islands and their position in genes. Common characteristics in the 100 nt sequences surrounding the epigenetic clock CpGs are G-quadruplexes and/or tentative splice site motifs. Median methylation increases significantly in sequences which adopt less stable structures during transcription. Methylation is higher when CpGs overlap with G-quadruplexes than when they precede them. Median methylation in epigenetic clock CpGs is higher in sequences expressed as single products rather than in multiple products and those containing single donors and multiple acceptors. Age-related methylation variation is significant in sequences without G-quadruplexes, particularly those producing low stability nascent RNA and those with splice sites. CpGs in sequences close to transcription start sites and those which are possibly never expressed (hypothetical proteins) undergo similar extent of age-related median methylation decrease and increase. Preservation of methylation is observed in CpG islands without G-quadruplexes, contrary to CpGs far from CpG islands (open sea). Sequences containing G-quadruplexes and RNA pseudoknots, determining the recognition by H3K27 histone methyltransferase, are hypomethylated. The presented structural DNA and co-transcriptional RNA analysis of epigenetic clock sequences, foreshadows the association of age-related methylation changes with the principle biological processes of DNA and histone methylation, splicing and chromatin silencing.
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Envelhecimento/genética , Relógios Biológicos , Metilação de DNA , Epigênese Genética , Ilhas de CpG , Quadruplex G , Humanos , Sítios de Splice de RNARESUMO
BACKGROUND: miR-29a is a small non-coding RNA that is known to repress collagen synthesis. Interestingly, elevated plasma miR-29a was reported to correlate with pronounced myocardial fibrosis in patients with hypertrophic cardiomyopathy. The objective of this study was to elucidate the origin of plasma miR-29a, and evaluate its significance as a biomarker. METHODS: miR-29a expression was evaluated in plasma (n=50) and myocardial samples (n=4) from patients with hypertrophic cardiomyopathy using RT-qPCR. RESULTS: Although miR-29a was highly expressed in the myocardium, miR-29a plasma levels did not show any correlation with serum troponin I levels (rs=-0.12, p=0.43), and the heart does not release significant amounts of miR-29a into the circulation via exosome secretion. Conversely, miR-29a was present in red blood cells, and plasma levels correlated significantly with markers of hemolysis: lactic dehydrogenase (rs=0.36, p=0.01) and the absorbance of oxyhemoglobin at 414nm (rs=0.39, p=0.006). Furthermore, the association between serum haptoglobin and the maximal blood flow velocity in the left ventricle outflow tract (rs=-0.42, p=0.008) indicated that intravascular hemolysis is a manifestation of the disease. CONCLUSIONS: miR-29a is highly expressed in myocardial tissue from patients with hypertrophic cardiomyopathy. In contrast, plasma miR-29a is primarily of nonmyocardial origin and is correlated significantly with the extent of hemolysis observed in these patients.
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Cardiomiopatia Hipertrófica/sangue , Hemólise , MicroRNAs/sangue , Adulto , Biomarcadores/sangue , Cardiomiopatia Hipertrófica/genética , Humanos , MicroRNAs/genética , Reação em Cadeia da Polimerase em Tempo RealRESUMO
Modification of p53 expression levels and its principle apoptosis and cell cycle regulatory partners, mouse double minute 2 homolog (MDM2) and p21, has been previously reported in various types of cancer. In the current study, the expression of Δ133p53 isoforms was investigated in lung carcinomas with respect to the expression of the aforementioned genes. The expression of p53 fulllength transcript and Δ133p53 isoforms α, ß and γ transcripts, MDM2 and p21 transcripts were determined by reverse transcriptionquantitative polymerase chain reaction, in total RNA isolated from 17 lung carcinoma specimens and 17 corresponding adjacent noncancerous tissues. RNA expression analysis was performed according to the Pfaffl equation and Rest tool using ßactin as a reference gene. Detection of the above proteins was additionally performed by western blotting. Significant overexpression of the Δ133p53 mRNAs was observed in cancerous as compared with adjacent noncancerous tissues (3.94fold), whereas fulllength p53 and MDM2 expression exhibited a smaller, however significant, increase. The expression of the p21 transcript was significantly reduced in cancerous specimens. Δ133p53 and p21 expression levels varied in parallel, however were not significantly correlated. p53 fulllength protein expression observed by western blot analysis strongly varied from the Δ133p53 isoforms, however MDM2 protein isoforms were not detectable and p21 protein was more abundant in noncancerous tissues. In conclusion, Δ133p53 mRNA levels is suggested as a potentially useful marker of malignancy in lung cancer. The absence of Δ133p53 protein in lung carcinomas, which overexpress Δ133p53 transcripts, may indicate the role of the latter in posttranscriptional regulation through RNA interference in the cell cycle and apoptosis.
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Inibidor de Quinase Dependente de Ciclina p21/genética , Regulação Neoplásica da Expressão Gênica , Neoplasias Pulmonares/genética , Proteína Supressora de Tumor p53/genética , Adulto , Idoso , Western Blotting , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Feminino , Humanos , Neoplasias Pulmonares/epidemiologia , Masculino , Pessoa de Meia-Idade , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas Proto-Oncogênicas c-mdm2/genética , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Fumar/genéticaRESUMO
Coding synonymous single nucleotide polymorphisms (SNPs) have attracted little attention until recently. However, such SNPs located in epigenetic, CpG sites modifying exonic splicing enhancers (ESEs) can be informative with regards to the recently verified association of intragenic methylation and splicing. The present study describes the association of type 2 diabetes (T2D) with the exonic, synonymous, epigenetic SNPs, rs3749166 in calpain 10 (CAPN10) glucose transporter (GLUT4) translocator and rs5404 in solute carrier family 2, member 2 (SLC2A2), also termed GLUT2, which, according to prior bioinformatic analysis, strongly modify the splicing potential of glucose transport-associated genes. Previous association studies reveal that only rs5404 exhibits a strong negative T2D association, while data on the CAPN10 polymorphism are contradictory. In the present study DNA from blood samples of 99 Greek non-diabetic control subjects and 71 T2D patients was analyzed. In addition, relevant publicly available cases (40) resulting from examination of 110 Personal Genome Project data files were analyzed. The frequency of the rs3749166 A allele, was similar in the patients and non-diabetic control subjects. However, AG heterozygotes were more frequent among patients (73.24% for Greek patients and 54.55% for corresponding non-diabetic control subjects; P=0.0262; total cases, 52.99 and 75.00%, respectively; P=0.0039). The rs5404 T allele was only observed in CT heterozygotes (Greek non-diabetic control subjects, 39.39% and Greek patients, 22.54%; P=0.0205; total cases, 34.69 and 21.28%, respectively; P=0.0258). Notably, only one genotype, heterozygous AG/CC, was T2D-associated (Greek non-diabetic control subjects, 29.29% and Greek patients, 56.33%; P=0.004; total cases, 32.84 and 56.58%, respectively; P=0.0008). Furthermore, AG/CC was strongly associated with very high (≥8.5%) glycosylated plasma hemoglobin levels among patients (P=0.0002 for all cases). These results reveal the complex heterozygotic SNP association with T2D, and indicate possible synergies of these epigenetic, splicing-regulatory, synonymous SNPs, which modify the splicing potential of two alternative glucose transport-associated genes.
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BACKGROUND: In the current study the presence of extracellular IL-1B, IL-8, OAZ and SAT mRNAs in the saliva was evaluated as a tool in the early detection of oral squamous cell carcinoma. METHODS: 34 patients with primary oral squamous cell carcinoma stage T1N0M0/T2N0M0, 20 patients with oral leukoplakia and dysplasia (15 patients with mild dysplasia and 5 with severe dysplasia/in situ carcinoma) and 31 matched healthy-control subjects were included in the study. The presence of IL-1B, IL-8, OAZ and SAT mRNA was evaluated in extracellular RNA isolated from saliva samples using sequence-specific primers and real-time RT-PCR. ROC curve analysis was used to estimate the ability of the biomarkers to detect oral squamous cell carcinoma patients. RESULTS: The data reveal that the combination of these four biomarkers provides a good predictive probability of up to 80% (AUC=0.799, p=0.002) for patients with oral squamous cell carcinoma but not patients suffering from oral leukoplakia with dysplasia. Moreover, the combination of only the two biomarkers (SAT and IL-8) also raises a high predictive ability of 75.5% (AUC=0.755, p=0.007) approximately equal to the four biomarkers suggesting the use of the two biomarkers only in the prediction model for oral squamous cell carcinoma patients limiting the economic and health cost in half. CONCLUSION: SAT and IL-8 mRNAs are present in the saliva in high quality and quantity, with a good discriminatory ability for oral squamous cell carcinoma patients only but not for patients with oral leukoplakia and dysplasia an oral potentially malignant disorder.
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Biomarcadores Tumorais/genética , Carcinoma de Células Escamosas/genética , Leucoplasia Oral/genética , Neoplasias Bucais/genética , RNA Mensageiro/metabolismo , Saliva/metabolismo , Carcinoma de Células Escamosas/patologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Neoplasias Bucais/patologiaRESUMO
Epigenetic changes include the addition of a methyl group to the 5' carbon of the cytosine ring, known as DNA methylation, which results in the generation of the fifth DNA base, namely 5-methylcytosine. During active or passive demethylation, an intermediate modified base is formed, 5-hydroxymethylcytosine. We have currently quantified 5-methylcytosine and 5-hydroxymethylcytosine in the liver and brain of mice treated with cocaine or heroin, using liquid chromatography/tandem mass spectrometry (LC-MS/MS). Our results show that global 5-methylcytosine levels are not affected by heroin or cocaine administration, neither in the liver nor in the brain. However, 5-hydroxymethylcytosine levels are reduced in the liver following cocaine administration, while they are not affected by cocaine in the brain or by heroin administration in the liver and the brain. Elucidation of the epigenetic phenomena that takes place with respect to drug abuse and addiction, via quantitative analysis of different modified bases, may enable a better understanding of the underlying mechanisms and may lead to more personalized and effective treatment options.
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5-Metilcitosina/análise , Cocaína/toxicidade , Citosina/análogos & derivados , Epigênese Genética/efeitos dos fármacos , Heroína/toxicidade , Animais , Encéfalo/efeitos dos fármacos , Química Encefálica/efeitos dos fármacos , Citosina/análise , Relação Dose-Resposta a Droga , Fígado/química , Fígado/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Epigenetic alterations, such as DNA methylation, are involved in the pathogenesis of various diseases, the toxicity of diverse agents, the process of aging, the development of stem cells and numerous other mechanisms. DNA methylation is one of the most well-studied epigenetic alterations in mammals. Nevertheless, the scientific interest is now focusing on novel modified nucleotides with potential regulatory roles, such as 5-hydroxymethylcytosine. We currently present and discuss novel bioanalytical strategies developed for the determination of various modified nucleotides in epigenetic studies.
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Metilação de DNA , DNA/química , Epigênese Genética , Nucleotídeos/análise , Nucleotídeos/genética , 5-Metilcitosina/análogos & derivados , Animais , Cromatografia Líquida de Alta Pressão/métodos , Citosina/análogos & derivados , Citosina/análise , DNA/genética , Humanos , Imuno-Histoquímica/métodos , Hibridização in Situ Fluorescente/métodosRESUMO
The identification of thousands of mutations yearly has put new challenges to researchers who are interested in fast and effective annotation as well as the prediction of potential implications to the gene regulation mechanisms. This work presents an integrative tool, called GREMET, for the prediction of alterations in gene splicing regulation inferred by mutations of the human genome. GREMET supports the characterization of mutations either single-point or indels with respect to their effect on the splicing potential of the neighboring sequences and the binding strength of auxiliary cis-acting splicing enhancers. In addition, GREMET identifies possible consequences of mutations on the DNA methylation through the disruption or creation of CpG sequences. Besides locus-specific mutations, GREMET performs the analyses on newly identified mutations and provides an easy-to-use Web interface helping researchers to save time in routine mutation analyses. GREMET is freely accessible at: http://kedip.med.auth.gr/biotools/gremet/.
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Análise Mutacional de DNA/estatística & dados numéricos , Regulação da Expressão Gênica , Mutação , Software , Acetil-CoA C-Acetiltransferase/deficiência , Acetil-CoA C-Acetiltransferase/genética , Ilhas de CpG , Metilação de DNA/genética , Genes p53 , Genoma Humano , Humanos , Mutação INDEL , Internet , Neoplasias/genética , Mutação Puntual , Sítios de Splice de RNA/genéticaRESUMO
Drug abuse is associated with epigenetic changes, such as histone modifications and DNA methylation. The purpose of the present study was to examine the effect of chronic cocaine and heroin administration on global DNA methylation in brain and liver. Male, 8 week old, C57BL/6J mice received heroin in a chronic 'intermittent' escalating dose paradigm, or cocaine in a chronic escalating dose 'binge' paradigm, which mimic the human pattern of opioid or cocaine abuse respectively. Following sacrifice, livers and brains were removed and DNA was extracted from them. The extracted DNA was hydrolyzed and 2'-deoxycytidine and 5-methyl-2'-deoxycytidine were determined by HPLC-UV. The % 5-methyl-2'-deoxycytidine content of DNA was significantly higher in the brain compared to the liver. There were no differences between the control animals and the cocaine or heroin treated animals in neither of the tissues examined, which is surprising since cocaine administration induced gross morphological changes in the liver. Moreover, there was no difference in the % 5-methyl-2'-deoxycytidine content of DNA between the cocaine and the heroin treated animals. The global DNA methylation status in the brain and liver of mice chronically treated with cocaine or heroin remains unaffected, but this finding cannot exclude the existence of anatomical region or gene-specific methylation differences. This is the first time that global DNA methylation in the liver and whole brain has been studied following chronic cocaine or heroin treatment.
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Encéfalo/efeitos dos fármacos , Transtornos Relacionados ao Uso de Cocaína/genética , Cocaína/toxicidade , Metilação de DNA/efeitos dos fármacos , Dependência de Heroína/genética , Heroína/toxicidade , Fígado/efeitos dos fármacos , Animais , Biomarcadores/metabolismo , Encéfalo/metabolismo , Encéfalo/patologia , Cromatografia Líquida de Alta Pressão , Transtornos Relacionados ao Uso de Cocaína/patologia , Desoxicitidina/análogos & derivados , Desoxicitidina/metabolismo , Modelos Animais de Doenças , Dependência de Heroína/patologia , Fígado/metabolismo , Fígado/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Espectrofotometria Ultravioleta , Fatores de TempoRESUMO
DNA methylation is presently accepted as a tentative regulatory parameter in splicing. Recently, we reported significant methylation differences among various exonic splicing-enhancing elements and alternative splicing events, based on CpG methylation data from the Human Epigenome Project for chromosomes 6, 20 and 22. Presently, using a different computational approach and the same database, we report: (a) significant increase of hypermethylation in intronic and exonic sequences close to acceptor sites, relative to overall introns and exons, respectively (1,973 CpGs examined); (b) frequent CpGs, mostly hypomethylated, in donors and infrequent CpGs mostly hypermethylated, in acceptors; and (c) hypermethylation in cassette exons which are occasionally spliced and have weaker average splicing potential, relative to constitutive exons (p < 0.0001). CpGs are hypomethylated in non-coding exons (only 16 % hypermethylation). Single-exon genes, similarly to first exons, frequently contain hypomethylated CpGs, while in internal and last exons CpGs are more frequently hypermethylated. Methylation is also more frequent in strange introns and splice sites processed by the minor spliceosome, e.g., ATAC, (p < 0.0001 in all cases), but not in sites of incomplete processing, e.g., retained introns or bleeding exons, (p = 0.706 and p = 0.313, respectively). Most Alus, which are known to contribute to transcript presentation, are heavily methylated, in contrast with other Alus, e.g., AluJo and mammalian interspersed repetitive elements which have been previously associated with alternative expression. These results elucidate the role of intragenic methylation in association with alternative splicing and facilitate the evaluation of genomic variations/polymorphisms and the development of tools for the prediction of alternative splicing events.
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Processamento Alternativo , Metilação de DNA , Sequências Repetitivas de Ácido Nucleico , Cromossomos Humanos Par 20 , Cromossomos Humanos Par 22 , Cromossomos Humanos Par 6 , Biologia Computacional/métodos , Humanos , Sítios de Splice de RNA , Transcrição GênicaRESUMO
BACKGROUND: Although the determination of 5-methyl-2'-deoxycytidine (5-MedC) in various biological samples is gaining increasing scientific interest, there are no data available regarding its stability. RESULTS: We have currently evaluated the stability of 5-MedC and 2'-deoxycytidine (dC) at -20°C, both in hydrolyzed and nonhydrolyzed calf thymus DNA (CT DNA), as well as following repetitive freeze-thaw cycles. HPLC-UV was used for the accurate determination of the two 2'-deoxynucleosides. Statistical evaluation of the results revealed that 5-MedC and dC were stable in hydrolyzed CT DNA for at least 7 days and in nonhydrolyzed CT DNA for at least 65 days, when these were stored at -20°C. Furthermore, both 2'-deoxynucleosides were stable for at least three repetitive freeze-thaw cycles. CONCLUSION: By using HPLC-UV, we have evaluated the stability of 5-MedC and dC under storage conditions and repetitive freeze-thaw cycles. Our results are informative about the way samples should be handled and stored in epigenetic studies.
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DNA/química , Desoxicitidina/análogos & derivados , Desoxicitidina/análise , Animais , Bovinos , Cromatografia Líquida de Alta Pressão/métodos , Metilação de DNA , Epigenômica , Congelamento , Humanos , Hidrólise , Análise de Regressão , Soluções , Manejo de Espécimes , Espectrofotometria UltravioletaRESUMO
The role of gene body methylation, which represents a major part of methylation in DNA, remains mostly unknown. Evidence based on the CpG distribution associates its presence with nucleosome positioning and alternative splicing. Recently, it was also shown that cytosine methylation influences splicing. However, to date, there is no methylation-based data on the association of methylation with alternative splicing and the distribution in exonic splicing enhancers (ESEs). We presently report that, based on the computational analysis of the Human Epigenome Project data, CpG hypermethylation (>80%) is frequent in alternatively spliced sites (particularly in noncanonical) but not in alternate promoters. The methylation frequency increases in sequences containing multiple putative ESEs. However, significant differences in the extent of methylation are observed among different ESEs. Specifically, moderate levels of methylation, ranging from 20% to 80%, are frequent in SRp55-binding elements, which are associated with response to extracellular conditions, but not in SF2/ASF, primarily responsible for alternative splicing, or in CpG islands. Finally, methylation is more frequent in the presence of AT repeats and CpGs separated by 10 nucleotides and lower in adjacent CpGs, probably indicating its dependence on helical formations and on the presence of nucleosome positioning-related sequences. In conclusion, our results show the regulation of methylation in ESEs and support its involvement in alternative splicing.
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Ilhas de CpG , Metilação de DNA , Elementos Facilitadores Genéticos , Sítios de Splice de RNA , Sequência de Bases , Bases de Dados de Ácidos Nucleicos , Repetições de Dinucleotídeos , Epigênese Genética , Éxons , Genoma Humano , HumanosRESUMO
The presence of extracellular circulating or cell-free RNA in biological fluids is becoming a promising diagnostic tool for non invasive and cost effective cancer detection. Extracellular RNA or miRNA as biological marker could be used either for the early detection and diagnosis of the disease or as a marker of recurrence patterns and surveillance. In this review article, we refer to the origin of the circulating extracellular RNA, we summarise the data on the biological fluids (serum/plasma, saliva, urine, cerebrospinal fluid and bronchial lavage fluid) of patients suffering from various types of malignancies reported to contain a substantial amount of circulating extracellular (or cell-free) RNAs and we discuss the appropriate reagents and methodologies needed to be employed in order to obtain RNA material of high quality and integrity for the majority of the experimental methods used in RNA expression analysis. Furthermore, we discuss the advantages and disadvantages of the RT-PCR or microarray methodology which are the methods more often employed in procedures of extracellular RNA analysis.
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Biomarcadores Tumorais/análise , Líquidos Corporais/química , RNA Neoplásico/isolamento & purificação , Biomarcadores Tumorais/genética , Humanos , RNA Neoplásico/análise , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
The true understanding of epigenetics evolved over time as our knowledge on DNA methylation and chromatin modifications and their effects on gene expression increased. The current flurry of research on epigenetics and the increasing documentation of the effects of various environmental factors on DNA methylation, chromatin modification, as well as on the expression of small non-coding RNAs (ncRNAs) have expanded the scope of research on the etiology of various diseases including cancer. The current review briefly discusses various molecular mechanisms of epigenetic regulation of gene expression, and expands the discussion with examples of heavy metal-induced alterations of gene expression and the associated epigenetic changes.
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Epigênese Genética , Metais Pesados/toxicidade , Animais , Metilação de DNA/efeitos dos fármacos , Metilação de DNA/genética , Epigênese Genética/efeitos dos fármacos , Histonas/genética , HumanosRESUMO
BACKGROUND: One of the principle symptoms of lead poisoning is the development of neurological disorders. Neuronal response is closely related to DNA methylation changes. Aim. In this study, we estimated p16 methylation in nine individuals exposed to lead using methylation-specific polymerase chain reaction followed by analysis of the methylated cytosine content of the product by thermal denaturation. RESULTS: We found that, based on lead blood concentration, lead-exposed individuals were divided into two groups. Among highly exposed individuals (blood Pb(2+) concentration = 51-100 microg/dL), we observed complete CpG methylation, whereas for low Pb(2+) concentrations (blood Pb(2+) concentration = 6-11 microg/dL), we observed partial methylation. CONCLUSION: Our results show that among lead-overexposed individuals, p16 methylation is frequent and extensive, and suggest that DNA methylation could be involved in the mechanism by which lead induces neurotoxicity.
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Metilação de DNA/efeitos dos fármacos , Genes p16/efeitos dos fármacos , Intoxicação do Sistema Nervoso por Chumbo em Adultos/fisiopatologia , Exposição Ocupacional/efeitos adversos , Regiões Promotoras Genéticas , Adulto , Estudos de Casos e Controles , Ilhas de CpG/efeitos dos fármacos , Citosina/metabolismo , Humanos , Chumbo/sangue , Chumbo/toxicidade , Intoxicação do Sistema Nervoso por Chumbo em Adultos/genética , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da PolimeraseRESUMO
Neuronal plasticity is associated with alternative splicing and epigenetic modulation. Recent evidence reveals the association of cytosine methylation with alternative splicing and splicing regulatory mechanisms. Single nucleotide polymorphisms (SNPs) are generally less frequent in conserved coding regions and probably in splice sites, compared to non-coding regions. CpG polymorphisms in coding regions and splice sites and their association with splicing regulatory elements have not been investigated till presently. We currently analyzed the CpG variability in 28 genes (361 constitutive and 105 alternative exons and the corresponding splice sites) associated with neurodegenerative diseases (ND). CpG polymorphisms in the splice sites of these genes are particularly frequent when compared to those at AG sequences. Moreover, in both constitutive and alternative exons, polymorphisms in CpGs are more frequent than in AG, GT sequences. On the contrary, in the polypyrimidine acceptor sequence C/T conservation is prominent indicating that in this locus the sequence of cytosines and thymines is preserved. Bioinformatic analysis of the splicing-associated regulatory elements in these exons and splice sites reveals that 18 out of a total of 39 SNPs which could strongly affect splicing (>1.5 score difference) contain CpG sequences. Cytosines are considerably more frequent and variable than expected at the position preceding the GT splice donors, while sites of epigenetic modification are absent from acceptors. The high CpG frequency in polymorphic splicing-associated sites implicates the involvement of epigenetic mechanisms in splicing selection decisions regulated by these sites, and indicates the complexity of genetic studies involving these, tentatively critical, polymorphisms in ND.