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Aims: Maternally inherited diabetes and deafness (MIDD) is a complex disorder characterized by multiorgan clinical manifestations, including diabetes, hearing loss, and ophthalmic complications. This pilot study aimed to elucidate the intricate interplay between nuclear and mitochondrial genetics, epigenetic modifications, and their potential implications in the pathogenesis of MIDD. Main methods: A comprehensive genomic approach was employed to analyze a Sicilian family affected by clinically characterized MIDD, negative to the only known causative m.3243 A > G variant, integrating whole-exome sequencing and whole-genome bisulfite sequencing of both nuclear and mitochondrial analyses. Key findings: Rare and deleterious variants were identified across multiple nuclear genes involved in retinal homeostasis, mitochondrial function, and epigenetic regulation, while complementary mitochondrial DNA analysis revealed a rich tapestry of genetic diversity across genes encoding components of the electron transport chain and ATP synthesis machinery. Epigenetic analyses uncovered significant differentially methylated regions across the genome and within the mitochondrial genome, suggesting a nuanced landscape of epigenetic modulation. Significance: The integration of genetic and epigenetic data highlighted the potential crosstalk between nuclear and mitochondrial regulation, with specific mtDNA variants influencing methylation patterns and potentially impacting the expression and regulation of mitochondrial genes. This pilot study provides valuable insights into the complex molecular mechanisms underlying MIDD, emphasizing the interplay between nucleus and mitochondrion, tracing the way for future research into targeted therapeutic interventions and personalized approaches for disease management.
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Arteriovenous malformation of the brain (bAVM) is a vascular phenotype related to brain defective angiogenesis. Involved vessels show impaired expression of vascular differentiation markers resulting in the arteriolar to venule direct shunt. In order to clarify aberrant gene expression occurring in bAVM, here we describe results obtained by methylome analysis performed on endothelial cells (ECs) isolated from bAVM specimens, compared to human cerebral microvascular ECs. Results were validated by quantitative methylation-specific PCR and quantitative realtime-PCR. Differential methylation events occur in genes already linked to bAVM onset, as RBPJ and KRAS. However, among differentially methylated genes, we identified EPHB1 and several other loci involved in EC adhesion as well as in EC/vascular smooth muscle cell (VSMC) crosstalk, suggesting that only endothelial dysfunction might not be sufficient to trigger the bAVM phenotype. Moreover, aberrant methylation pattern was reported for many lncRNA genes targeting transcription factors expressed during neurovascular development. Among these, the YBX1 that was recently shown to target the arteridin coding gene. Finally, in addition to the conventional CpG methylation, we further considered the role of impaired CHG methylation, mainly occurring in brain at embryo stage. We showed as differentially CHG methylated genes are clustered in pathways related to EC homeostasis, as well as to VSMC-EC crosstalk, suggesting as impairment of this interaction plays a prominent role in loss of vascular differentiation, in bAVM phenotype.
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Aims: This pilot study investigates the potential pathogenic role of G-quadruplex (G4) structures in RPGR-associated retinal degeneration, starting from a case of suspected X-linked form affected family. We hypothesize that the stabilization of these structures might alter DNA replication and transcription, inducing genetic instability and influencing gene expression. Main methods: We conducted whole genome amplification experiments and next-generation sequencing to detect the blockade of polymerase activity by G4 structures. Our specific focus was the RPGR gene, which hosts a high concentration of predicted G4-forming motifs and is implicated in most X-linked retinal degeneration cases. To understand the potential interference of G4 structures, we applied computational and 3D molecular modeling to visualize interferences in DNA replication and transcription regulation. Key findings: Our data confirmed the obstruction of DNA polymerase enzymes by G4 structures, particularly when stabilized by the compound pyridostatin. This obstruction was evident in the reduced amplification of RPGR gene regions and a shift in the start/end sites of putative G4 motifs. Moreover, the modeling indicated a potential disruption of critical promoter elements and RNA polymerase binding, which could drastically alter gene expression. Significance: Our findings suggest that G4 formation in the RPGR gene could lead to genetic instability and affect the expression of RPGR, contributing to retinal dystrophy. Moreover, this study underscores the broader implications of G4 structures in other genetic disorders. Improved understanding of G4 structures could reveal novel therapeutic targets to combat genetic disorders, promoting the advancement of personalized medicine and precision health.
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Beyond their crucial role in energy production, mitochondria harbor a distinct genome subject to epigenetic regulation akin to that of nuclear DNA. This paper delves into the nascent but rapidly evolving fields of mitoepigenetics and mitoepigenomics, exploring the sophisticated regulatory mechanisms governing mitochondrial DNA (mtDNA). These mechanisms encompass mtDNA methylation, the influence of non-coding RNAs (ncRNAs), and post-translational modifications of mitochondrial proteins. Together, these epigenetic modifications meticulously coordinate mitochondrial gene transcription, replication, and metabolism, thereby calibrating mitochondrial function in response to the dynamic interplay of intracellular needs and environmental stimuli. Notably, the dysregulation of mitoepigenetic pathways is increasingly implicated in mitochondrial dysfunction and a spectrum of human pathologies, including neurodegenerative diseases, cancer, metabolic disorders, and cardiovascular conditions. This comprehensive review synthesizes the current state of knowledge, emphasizing recent breakthroughs and innovations in the field. It discusses the potential of high-resolution mitochondrial epigenome mapping, the diagnostic and prognostic utility of blood or tissue mtDNA epigenetic markers, and the promising horizon of mitochondrial epigenetic drugs. Furthermore, it explores the transformative potential of mitoepigenetics and mitoepigenomics in precision medicine. Exploiting a theragnostic approach to maintaining mitochondrial allostasis, this paper underscores the pivotal role of mitochondrial epigenetics in charting new frontiers in medical science.
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Metilação de DNA , DNA Mitocondrial , Epigênese Genética , Mitocôndrias , Humanos , Mitocôndrias/genética , Mitocôndrias/metabolismo , Mitocôndrias/patologia , DNA Mitocondrial/genética , DNA Mitocondrial/metabolismo , Processamento de Proteína Pós-Traducional/genética , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Doenças Mitocondriais/genética , Doenças Mitocondriais/metabolismo , Doenças Mitocondriais/patologia , RNA não Traduzido/genética , RNA não Traduzido/metabolismo , Doenças Neurodegenerativas/genética , Doenças Neurodegenerativas/metabolismo , Doenças Neurodegenerativas/patologia , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patologiaRESUMO
OBJECTIVES: To assess effectiveness of an experimental sterilization method based on the exposure of an O3/O2 gas mixture directly inside the packaging for clear aligners. MATERIALS AND METHODS: Fifty samples consisting of pieces of polyethylene terephthalate glycol (PET-G) aligners were contaminated by manual handling and subsequently divided into different groups (n = 30 for exposure to O3/O2 gas at different times, n = 10 for positive control with 2% chlorhexidine digluconate, n = 10 for negative control). The measurement of optical densities (OD) of the initial and final microbial cultures was recorded for all groups. Kruskal-Wallis test was used for differences between groups while Wilcoxon test was used to compare initial and final OD values within groups. Statistical significance was set at P < .05. RESULTS: Comparison within the groups showed statistically significant differences for exposure to the gaseous mixture (72 hours), for positive and negative controls. Other significant differences were found in the multiple comparisons between the application of gaseous ozone (48 hours and 72 hours) and the negative control. CONCLUSIONS: The direct exposure of gaseous ozone on the aligners inside their packaging showed microbicidal capacity at 72 hours, which was equivalent to the positive control with immersion in chlorhexidine digluconate. This innovative sterilization procedure could be considered in the final manufacturing processes of clear aligners to eliminate the potentially pathogenic microorganisms that are deposited on surfaces of these orthodontic devices.
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Clorexidina/análogos & derivados , Aparelhos Ortodônticos Removíveis , Ozônio , Clorexidina/farmacologia , EsterilizaçãoRESUMO
In the early stages of Alzheimer-Perusini's disease (AD), individuals often experience vision-related issues such as color vision impairment, reduced contrast sensitivity, and visual acuity problems. As the disease progresses, there is a connection with glaucoma and age-related macular degeneration (AMD) leading to retinal cell death. The retina's involvement suggests a link with the hippocampus, where most AD forms start. A thinning of the retinal nerve fiber layer (RNFL) due to the loss of retinal ganglion cells (RGCs) is seen as a potential AD diagnostic marker using electroretinography (ERG) and optical coherence tomography (OCT). Amyloid beta fragments (Aß), found in the eye's vitreous and aqueous humor, are also present in the cerebrospinal fluid (CSF) and accumulate in the retina. Aß is known to cause tau hyperphosphorylation, leading to its buildup in various retinal layers. However, diseases like AD are now seen as mixed proteinopathies, with deposits of the prion protein (PrP) and α-synuclein found in affected brains and retinas. Glial cells, especially microglial cells, play a crucial role in these diseases, maintaining immunoproteostasis. Studies have shown similarities between retinal and brain microglia in terms of transcription factor expression and morphotypes. All these findings constitute a good start to achieving better comprehension of neurodegeneration in both the eye and the brain. New insights will be able to bring the scientific community closer to specific disease-modifying therapies.
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In recent years, mesenchymal stem cells (MSC) have been considered the most effective source for regenerative medicine, especially due to released soluble paracrine bioactive components and extracellular vesicles. These factors, collectively called the secretome, play crucial roles in immunomodulation and in improving survival and regeneration capabilities of injured tissue. Recently, there has been a growing interest in the secretome released by retinal cytotypes, especially retinal pigment epithelium and Müller glia cells. The latter trophic factors represent the key to preserving morphofunctional integrity of the retina, regulating biological pathways involved in survival, function and responding to injury. Furthermore, these factors can play a pivotal role in onset and progression of retinal diseases after damage of cell secretory function. In this review, we delineated the importance of cross-talk between MSCs and retinal cells, focusing on common/induced secreted factors, during experimental therapy for retinal diseases. The cross-link between the MSC and retinal cell secretomes suggests that the MSC secretome can modulate the retinal cell secretome and vice versa. For example, the MSC secretome can protect retinal cells from degeneration by reducing oxidative stress, autophagy and programmed cell death. Conversely, the retinal cell secretome can influence the MSC secretome by inducing changes in MSC gene expression and phenotype.
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Alzheimer-Perusini's (AD) disease represents the most spread dementia around the world and constitutes a serious problem for public health. It was first described by the two physicians from whom it took its name. Nowadays, we have extensively expanded our knowledge about this disease. Starting from a merely clinical and histopathologic description, we have now reached better molecular comprehension. For instance, we passed from an old conceptualization of the disease based on plaques and tangles to a more modern vision of mixed proteinopathy in a one-to-one relationship with an alteration of specific glial and neuronal phenotypes. However, no disease-modifying therapies are yet available. It is likely that the only way to find a few "magic bullets" is to deepen this aspect more and more until we are able to draw up specific molecular profiles for single AD cases. This review reports the most recent classifications of AD atypical variants in order to summarize all the clinical evidence using several discrimina (for example, post mortem neurofibrillary tangle density, cerebral atrophy, or FDG-PET studies). The better defined four atypical forms are posterior cortical atrophy (PCA), logopenic variant of primary progressive aphasia (LvPPA), behavioral/dysexecutive variant and AD with corticobasal degeneration (CBS). Moreover, we discuss the usefulness of such classifications before outlining the molecular-genetic aspects focusing on microglial activity or, more generally, immune system control of neuroinflammation and neurodegeneration.
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In the central nervous system, thrombin-mediated activation of protease-activated receptors (PARs) results in neuroinflammation and increased vascular permeability. These events have been linked to cancer and neurodegeneration. Endothelial cells (ECs) isolated from sporadic cerebral cavernous malformation (CCM) specimens showed dysregulation of genes involved in "thrombin-mediated PAR-1 activation" signaling. CCM is a vascular disease involving brain capillaries. In CCM, ECs show defective cell junctions. Oxidative stress and neuroinflammation play a key role in disease onset and progression. In order to confirm the possible role of thrombin pathway in sporadic CCM pathogenesis, we evaluated PARs expression in CCM-ECs. We found that sporadic CCM-ECs overexpress PAR1, PAR3 and PAR4, together with other coagulation factor encoding genes. Moreover, we investigated about expression of the three familial CCM genes (KRIT1, CCM2 and PDCD10) in human cerebral microvascular ECs, following thrombin exposure, as well as protein level. Thrombin exposure affects EC viability and results in dysregulation of CCM gene expression and, then, in decreased protein level. Our results confirm amplification of PAR pathway in CCM suggesting, for the first time, the possible role of PAR1-mediated thrombin signaling in sporadic CCM. Thrombin-mediated PARs over activation results in increased blood-brain barrier permeability due to loss of cell junction integrity and, in this context, also the three familial CCM genes may be involved.
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Hemangioma Cavernoso do Sistema Nervoso Central , Humanos , Hemangioma Cavernoso do Sistema Nervoso Central/genética , Hemangioma Cavernoso do Sistema Nervoso Central/metabolismo , Hemangioma Cavernoso do Sistema Nervoso Central/patologia , Células Endoteliais/metabolismo , Doenças Neuroinflamatórias , Receptor PAR-1/genética , Receptor PAR-1/metabolismo , Trombina/farmacologia , Proteínas Proto-Oncogênicas/genéticaRESUMO
Cone-rod dystrophies (CORDs) are a heterogeneous group of inherited retinopathies (IRDs) with more than 30 already known disease-causing genes. Uncertain phenotypes and extended range of intra- and interfamilial heterogenicity make still difficult to determine a precise genotype-phenotype correlation. Here, we used a next-generation sequencing approach to study a Sicilian family with a suspected form of CORD. Affected family members underwent ophthalmological examinations and a proband, blind from 50 years, underwent whole genome and exome sequencing. Variant analysis was enriched by pathway analysis and relevant variants were, then, investigated in other family members and in 100 healthy controls from Messina. CORD diagnosis with an intricate pattern of symptoms was confirmed by ophthalmological examinations. A total of about 50,000 variants were identified in both proband's genome and exome. All affected family members presented specific genotypes mainly determined by mutated GUCY2D gene, and different phenotypical traits, mainly related to focus and color perception. Thus, we looked for possible modifier genes. According to relationship with GUCY2D, predicted functional effects, eye localization, and ocular disease affinity, only 9 variants, carried by 6 genes (CACNG8, PAX2, RXRG, CCDC175, PDE4DIP and LTF), survived the filtering. These genes encode key proteins involved in cone development and survival, and retina neurotransmission. Among analyzed variants, CACNG8c.*6819A>T and the new CCDC175 c.76C>T showed extremely low frequency in the control group, suggesting a key role on disease phenotypes. Such discovery could enforce the role of modifier genes into CORD onset/progression, contributing to improve diagnostic test towards a better personalized medicine.
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Distrofias de Cones e Bastonetes , Retinose Pigmentar , Humanos , Canais de Cálcio/genética , Distrofias de Cones e Bastonetes/genética , Análise Mutacional de DNA , Genes Modificadores , Mutação , Linhagem , Fenótipo , Projetos Piloto , Retinose Pigmentar/genéticaRESUMO
Oxidative stress represents one of the principal causes of inherited retinal dystrophies, with many related molecular mechanisms still unknown. We investigated the posttranscriptional RNA editing landscape of human retinal pigment epithelium cells (RPE) exposed to the oxidant agent N-retinylidene-N-retinyl ethanolamine (A2E) for 1 h, 2 h, 3 h and 6 h. Using a transcriptomic approach, refined with a specific multialgorithm pipeline, 62,880 already annotated and de novo RNA editing sites within about 3000 genes were identified among all samples. Approximately 19% of these RNA editing sites were found within 3' UTR, including sites common to all time points that were predicted to change the binding capacity of 359 miRNAs towards 9654 target genes. A2E exposure also determined significant gene expression differences in deaminase family ADAR, APOBEC and ADAT members, involved in canonical and tRNA editing events. On GO and KEGG enrichment analyses, genes that showed different RNA editing levels are mainly involved in pathways strongly linked to a possible neovascularization of retinal tissue, with induced apoptosis mediated by the ECM and surface protein altered signaling. Collectively, this work demonstrated dynamic RNA editome profiles in RPE cells for the first time and shed more light on new mechanisms at the basis of retinal degeneration.
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Novel insights on regulation of gene expression mechanisms highlight the pivotal role of epitranscriptomic modifications on decision about transcript fate. These modifications include methylation of adenosine and cytosine in RNA molecules. Impairment of the normal epitranscriptome profile was observed in several pathological conditions, such as cancer and neurodegeneration. However, it is still unknown if alteration of this regulatory mechanism can be involved in cerebral cavernous malformation (CCM) development. CCM is a rare genetic condition affecting brain microvasculature, resulting from mutations in the three genes KRIT1, CCM2, and PDCD10. By data integration of association study, in silico prediction, and gene expression analysis, we evaluated role of single nucleotide polymorphisms (SNPs) highly recurrent in patients with CCM, on CCM gene expression regulation. Results showed that several of these SNPs lead to a drastic downexpression, in KRIT1 and CCM2 genes and this downregulation can be due to alteration of epitranscriptome profile, occurring these SNPs in gene regions that are subject to epitranscriptome modifications. These data suggest that this novel mechanism of gene expression regulation can be consider to further investigation on CCM pathogenesis.
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Hemangioma Cavernoso do Sistema Nervoso Central , Adenosina/genética , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Proteínas de Transporte/metabolismo , Citosina , Hemangioma Cavernoso do Sistema Nervoso Central/genética , Hemangioma Cavernoso do Sistema Nervoso Central/metabolismo , Humanos , Proteínas de Membrana/metabolismo , Polimorfismo de Nucleotídeo Único/genética , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , RNA Mensageiro/genéticaRESUMO
By regulating several phases of gene expression, RNA editing modifications contribute to maintaining physiological RNA expression levels. RNA editing dysregulation can affect RNA molecule half-life, coding/noncoding RNA interaction, alternative splicing, and circular RNA biogenesis. Impaired RNA editing has been observed in several pathological conditions, including cancer and Alzheimer's disease. No data has been published yet on the editome profile of endothelial cells (ECs) isolated from human cerebral cavernous malformation (CCM) lesions. Here, we describe a landscape of editome modifications in sporadic CCM-derived ECs (CCM-ECs) by comparing editing events with those observed in human brain microvascular endothelial cells (HBMECs). With a whole transcriptome-based variant calling pipeline, we identified differential edited genes in CCM-ECs that were enriched in pathways related to angiogenesis, apoptosis and cell survival, inflammation and, in particular, to thrombin signalling mediated by protease-activated receptors and non-canonical Wnt signalling. These pathways, not yet associated to CCM development, could be a novel field for further investigations on CCM molecular mechanisms. Moreover, enrichment analysis of differentially edited miRNAs suggested additional small noncoding transcripts to consider for development of targeted therapies.
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Hemangioma Cavernoso do Sistema Nervoso Central , MicroRNAs , Células Endoteliais/metabolismo , Hemangioma Cavernoso do Sistema Nervoso Central/genética , Hemangioma Cavernoso do Sistema Nervoso Central/metabolismo , Hemangioma Cavernoso do Sistema Nervoso Central/patologia , Humanos , MicroRNAs/metabolismo , Neovascularização Patológica/genética , TranscriptomaRESUMO
BACKGROUND: Trimethylaminuria (TMAU) is a rare genetic disease characterized by the accumulation of trimethylamine (TMA) and its subsequent excretion trough main body fluids, determining the characteristic fish odour in affected patients. We realized an experimental study to investigate the role of several coding variants in the causative gene FMO3, that were only considered as polymorphic or benign, even if the available literature on them did not functionally explain their ineffectiveness on the encoded enzyme. METHODS: Mutational analysis of 26 TMAU patients was realized by Sanger sequencing. Detected variants were, subsequently, deeply statistically and in silico characterized to determine their possible effects on the enzyme activity. To achieve this goal, a docking prediction for TMA/FMO3 and an unbinding pathway study were performed. Finally, a TMAO/TMA urine quantification by 1H-NMR spectroscopy was performed to support modelling results. RESULTS: The FMO3 screening of all patients highlighted the presence of 17 variants distributed in 26 different haplotypes. Both non-sense and missense considered variants might impair the enzymatic kinetics of FMO3, probably reducing the interaction time between the protein catalytic site and TMA, or losing the wild-type binding site. CONCLUSIONS: Even if further functional assays will confirm our predictive results, considering the possible role of FMO3 variants with still uncertain effects, might be a relevant step towards the detection of novel scenarios in TMAU etiopathogenesis.
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Erros Inatos do Metabolismo , Metilaminas/urina , Modelos Moleculares , Mutação , Oxigenases , Adulto , Feminino , Humanos , Masculino , Erros Inatos do Metabolismo/enzimologia , Erros Inatos do Metabolismo/genética , Erros Inatos do Metabolismo/urina , Ressonância Magnética Nuclear Biomolecular , Oxigenases/química , Oxigenases/genética , Oxigenases/metabolismoRESUMO
The neurovascular unit (NVU) is a relatively recent concept that clearly describes the relationship between brain cells and their blood vessels. The components of the NVU, comprising different types of cells, are so interrelated and associated with each other that they are considered as a single functioning unit. For this reason, even slight disturbances in the NVU could severely affect brain homeostasis and health. In this review, we aim to describe the current state of knowledge concerning the role of oxidative stress on the neurovascular unit and the role of a single cell type in the NVU crosstalk.
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During the last (15) years, improved omics sequencing technologies have expanded the scale and resolution of various biological applications, generating high-throughput datasets that require carefully chosen software tools to be processed. Therefore, following the sequencing development, bioinformatics researchers have been challenged to implement alignment algorithms for next-generation sequencing reads. However, nowadays selection of aligners based on genome characteristics is poorly studied, so our benchmarking study extended the "state of art" comparing 17 different aligners. The chosen tools were assessed on empirical human DNA- and RNA-Seq data, as well as on simulated datasets in human and mouse, evaluating a set of parameters previously not considered in such kind of benchmarks. As expected, we found that each tool was the best in specific conditions. For Ion Torrent single-end RNA-Seq samples, the most suitable aligners were CLC and BWA-MEM, which reached the best results in terms of efficiency, accuracy, duplication rate, saturation profile and running time. About Illumina paired-end osteomyelitis transcriptomics data, instead, the best performer algorithm, together with the already cited CLC, resulted Novoalign, which excelled in accuracy and saturation analyses. Segemehl and DNASTAR performed the best on both DNA-Seq data, with Segemehl particularly suitable for exome data. In conclusion, our study could guide users in the selection of a suitable aligner based on genome and transcriptome characteristics. However, several other aspects, emerged from our work, should be considered in the evolution of alignment research area, such as the involvement of artificial intelligence to support cloud computing and mapping to multiple genomes. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s00521-021-06188-z.
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Blindness due to photoreceptor degeneration is observed in both genetic and acquired eye disorders. Long blue light exposure can contribute to increase levels of oxidative compounds within the retinal pigment epithelium (RPE), enhancing risk of retinal damage. In retina, reactive oxygen species contribute to the activation of inflammatory cascade. If chronic, this inflammatory response can result in photoreceptor death. Therefore, we investigated the effects of the endogenous adduct N-retinylidene-N-retinylethanolamine (A2E) on RPE cells, in order to identify the most dysregulated cytokines and their related inflammatory pathways. RPE cells were exposed to A2E and blue light for 3h and 6h. By transcriptome analysis, we identified differentially expressed genes in A2E-treated cells, when compared to untreated ones. Expression values were quantified by the Limma R package. Enrichment analysis was performed according to the "Reactome" and the Gene Ontology databases. Expression of pro-inflammatory cytokines increased after 3h of A2E treatment and pathways related to IL-6 and IL-1 signaling resulted enriched. Also the up-regulation of genes having a protective role against inflammation was observed. Moreover, our results show that ferroptosis could contribute to RPE degeneration induced by A2E and blue light. Dysregulated genes related to retinal degeneration triggered by oxidative damage and inflammatory response activation identified in this study can be considered as potential biomarkers for targeted therapies.