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1.
Front Genet ; 13: 1042483, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36468035

RESUMO

Multiple Sclerosis (MS) is an autoimmune, neurological disease, commonly presenting with a relapsing-remitting form, that later converts to a secondary progressive stage, referred to as RRMS and SPMS, respectively. Early treatment slows disease progression, hence, accurate and early diagnosis is crucial. Recent advances in large-scale data processing and analysis have progressed molecular biomarker development. Here, we focus on small RNA data derived from cell-free cerebrospinal fluid (CSF), cerebrospinal fluid cells, plasma and peripheral blood mononuclear cells as well as CSF cell methylome data, from people with RRMS (n = 20), clinically/radiologically isolated syndrome (CIS/RIS, n = 2) and neurological disease controls (n = 14). We applied multiple co-inertia analysis (MCIA), an unsupervised and thereby unbiased, multivariate method for simultaneous data integration and found that the top latent variable classifies RRMS status with an Area Under the Receiver Operating Characteristics (AUROC) score of 0.82. Variable selection based on Lasso regression reduced features to 44, derived from the small RNAs from plasma (20), CSF cells (8) and cell-free CSF (16), with a marginal reduction in AUROC to 0.79. Samples from SPMS patients (n = 6) were subsequently projected on the latent space and differed significantly from RRMS and controls. On contrary, we found no differences between relapse and remission or between inflammatory and non-inflammatory disease controls, suggesting that the latent variable is not prone to inflammatory signals alone, but could be MS-specific. Hence, we here showcase that integration of small RNAs from plasma and CSF can be utilized to distinguish RRMS from SPMS and neurological disease controls.

2.
Epigenetics ; 17(10): 1195-1204, 2022 10.
Artigo em Inglês | MEDLINE | ID: mdl-34709110

RESUMO

DNA methylation is the most studied epigenetic mark involved in regulation of gene expression. For low input samples, a limited number of methods for quantifying DNA methylation genome-wide has been evaluated. Here, we compared a series of input DNA amounts (1-10ng) from two methylome library preparation protocols, enzymatic methyl-seq (EM-seq) and post-bisulfite adaptor tagging (PBAT) adapted from single-cell PBAT. EM-seq takes advantage of enzymatic activity while PBAT relies on conventional bisulfite conversion for detection of DNA methylation. We found that both methods accurately quantified DNA methylation genome-wide. They produced expected distribution patterns around genomic features, high C-T transition efficiency at non-CpG sites and high correlation between input amounts. However, EM-seq performed better in regard to library and sequencing quality, i.e. EM-seq produced larger insert sizes, higher alignment rates and higher library complexity with lower duplication rate compared to PBAT. Moreover, EM-seq demonstrated higher CpG coverage, better CpG site overlap and higher consistency between input series. In summary, our data suggests that EM-seq overall performed better than PBAT in whole-genome methylation quantification of low input samples.


Assuntos
Metilação de DNA , Epigenoma , Ilhas de CpG , DNA/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Análise de Sequência de DNA/métodos , Sulfitos
3.
Proc Natl Acad Sci U S A ; 118(17)2021 04 27.
Artigo em Inglês | MEDLINE | ID: mdl-33879606

RESUMO

Multiple sclerosis (MS) is a chronic inflammatory demyelinating disease affecting the central nervous system (CNS). Small non-coding RNAs (sncRNAs) and, in particular, microRNAs (miRNAs) have frequently been associated with MS. Here, we performed a comprehensive analysis of all classes of sncRNAs in matching samples of peripheral blood mononuclear cells (PBMCs), plasma, cerebrospinal fluid (CSF) cells, and cell-free CSF from relapsing-remitting (RRMS, n = 12 in relapse and n = 11 in remission) patients, secondary progressive (SPMS, n = 6) MS patients, and noninflammatory and inflammatory neurological disease controls (NINDC, n = 11; INDC, n = 5). We show widespread changes in miRNAs and sncRNA-derived fragments of small nuclear, nucleolar, and transfer RNAs. In CSF cells, 133 out of 133 and 115 out of 117 differentially expressed sncRNAs were increased in RRMS relapse compared to remission and RRMS compared to NINDC, respectively. In contrast, 65 out of 67 differentially expressed PBMC sncRNAs were decreased in RRMS compared to NINDC. The striking contrast between the periphery and CNS suggests that sncRNA-mediated mechanisms, including alternative splicing, RNA degradation, and mRNA translation, regulate the transcriptome of pathogenic cells primarily in the CNS target organ.


Assuntos
Esclerose Múltipla/genética , Esclerose Múltipla/imunologia , Transcriptoma/genética , Adulto , Feminino , Expressão Gênica/genética , Perfilação da Expressão Gênica/métodos , Humanos , Leucócitos/metabolismo , Leucócitos Mononucleares/metabolismo , Masculino , MicroRNAs/sangue , MicroRNAs/líquido cefalorraquidiano , MicroRNAs/genética , Pessoa de Meia-Idade , Esclerose Múltipla/metabolismo , Esclerose Múltipla Crônica Progressiva/genética , Esclerose Múltipla Recidivante-Remitente/genética , Recidiva Local de Neoplasia/metabolismo , Pequeno RNA não Traduzido/sangue , Pequeno RNA não Traduzido/líquido cefalorraquidiano , Pequeno RNA não Traduzido/genética
4.
J Autoimmun ; 101: 17-25, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31014917

RESUMO

Multiple sclerosis (MS) is a leading cause of progressive disability among young adults caused by inflammation, demyelination and axonal loss in the central nervous system. Small non-coding RNAs (sncRNAs) are important regulators of various biological processes and could therefore play important roles in MS. Over the past decade, a large number of studies investigated sncRNAs in MS patients, focusing primarily on microRNAs (miRNAs). Overwhelming 500 miRNAs have been reported as dysregulated in MS. Nevertheless, owing to a large heterogeneity between studies it is challenging to evaluate the reproducibility of findings, in turn hampering our knowledge about the functional roles of miRNAs in disease. We systematically searched main databases and evaluated results from all studies that examined sncRNAs in MS to date (n = 61) and provided a detailed overview of experimental design and findings of these studies. We focused on the mechanisms of the most dysregulated sncRNAs and used predicted targets of the most dysregulated sncRNAs as input for functional enrichment analysis to highlight affected pathways. The prime affected pathway was TGF-ß signaling. This multifunctional cytokine is important in the differentiation and function of T helper type 17 (Th17) and regulatory T (Treg) cells, with opposing functions in the disease. Recent studies demonstrate the importance of miRNAs in controlling the balance between Th17/Th1 cells and Tregs and, importantly, the potential to exploit this paradigm for therapeutic purposes. Additionally, some of the discussed miRNAs could potentially serve as biomarkers of disease. In order to assist researchers in evaluating the evidence of a particular sncRNA in the pathogenesis of MS, we provide a detailed overview of experimental design and findings of these studies to date.


Assuntos
Biomarcadores , Predisposição Genética para Doença , Terapia Genética , Terapia de Alvo Molecular , Esclerose Múltipla/genética , Esclerose Múltipla/terapia , Pequeno RNA não Traduzido , Animais , MicroRNA Circulante , Regulação da Expressão Gênica , Redes Reguladoras de Genes , Estudos de Associação Genética , Humanos , MicroRNAs/genética , Terapia de Alvo Molecular/métodos , Esclerose Múltipla/diagnóstico , Interferência de RNA
5.
Curr Genomics ; 19(5): 339-355, 2018 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-30065610

RESUMO

Spinal Muscular Atrophy (SMA) is a neuromuscular disorder caused by mutations in the SMN1 gene. Being a monogenic disease, it is characterized by high clinical heterogeneity. Variations in penetrance and severity of symptoms, as well as clinical discrepancies between affected family members can result from modifier genes influence on disease manifestation. SMN2 gene copy number is known to be the main phenotype modifier and there is growing evidence of additional factors contributing to SMA severity. Potential modifiers of spinal muscular atrophy can be found among the wide variety of different factors, such as multiple proteins interacting with SMN or promoting motor neuron survival, epigenetic modifications, transcriptional or splicing factors influencing SMN2 expression. Study of these factors enables to reveal mechanisms underlying SMA pathology and can have pronounced clinical application.

6.
Nat Commun ; 9(1): 2397, 2018 06 19.
Artigo em Inglês | MEDLINE | ID: mdl-29921915

RESUMO

The human leukocyte antigen (HLA) haplotype DRB1*15:01 is the major risk factor for multiple sclerosis (MS). Here, we find that DRB1*15:01 is hypomethylated and predominantly expressed in monocytes among carriers of DRB1*15:01. A differentially methylated region (DMR) encompassing HLA-DRB1 exon 2 is particularly affected and displays methylation-sensitive regulatory properties in vitro. Causal inference and Mendelian randomization provide evidence that HLA variants mediate risk for MS via changes in the HLA-DRB1 DMR that modify HLA-DRB1 expression. Meta-analysis of 14,259 cases and 171,347 controls confirms that these variants confer risk from DRB1*15:01 and also identifies a protective variant (rs9267649, p < 3.32 × 10-8, odds ratio = 0.86) after conditioning for all MS-associated variants in the region. rs9267649 is associated with increased DNA methylation at the HLA-DRB1 DMR and reduced expression of HLA-DRB1, suggesting a modulation of the DRB1*15:01 effect. Our integrative approach provides insights into the molecular mechanisms of MS susceptibility and suggests putative therapeutic strategies targeting a methylation-mediated regulation of the major risk gene.


Assuntos
Metilação de DNA , Predisposição Genética para Doença/genética , Cadeias HLA-DRB1/genética , Esclerose Múltipla/genética , Polimorfismo de Nucleotídeo Único , Adulto , Idoso , Células Cultivadas , Estudos de Coortes , Feminino , Regulação da Expressão Gênica , Humanos , Masculino , Metanálise como Assunto , Pessoa de Meia-Idade , Esclerose Múltipla/imunologia , Esclerose Múltipla/patologia , Fatores de Risco , Adulto Jovem
7.
Physiol Genomics ; 49(9): 447-461, 2017 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-28754822

RESUMO

Multiple sclerosis (MS) is a chronic inflammatory and demyelinating disease of the central nervous system. MS likely results from a complex interplay between predisposing causal gene variants (the strongest influence coming from HLA class II locus) and environmental risk factors such as smoking, infectious mononucleosis, and lack of sun exposure/vitamin D. However, little is known about the mechanisms underlying MS development and progression. Moreover, the clinical heterogeneity and variable response to treatment represent additional challenges to a comprehensive understanding and efficient treatment of disease. Epigenetic processes, such as DNA methylation and histone posttranslational modifications, integrate influences from the genes and the environment to regulate gene expression accordingly. Studying epigenetic modifications, which are stable and reversible, may provide an alternative approach to better understand and manage disease. We here aim to review findings from epigenetic studies in MS and further discuss the challenges and clinical opportunities arising from epigenetic research, many of which apply to other diseases with similar complex etiology. A growing body of evidence supports a role of epigenetic processes in the mechanisms underlying immune pathogenesis and nervous system dysfunction in MS. However, disparities between studies shed light on the need to consider possible confounders and methodological limitations for a better interpretation of the data. Nevertheless, translational use of epigenetics might offer new opportunities in epigenetic-based diagnostics and therapeutic tools for a personalized care of MS patients.


Assuntos
Pesquisa Biomédica , Epigênese Genética , Esclerose Múltipla/genética , Animais , Biomarcadores/metabolismo , Encéfalo/metabolismo , Encéfalo/patologia , Humanos
8.
Behav Brain Funct ; 12(1): 17, 2016 Jun 06.
Artigo em Inglês | MEDLINE | ID: mdl-27267954

RESUMO

Brain-derived neurotrophic factor (BDNF) plays an important role in nervous system development and function and it is well established that BDNF is involved in the pathogenesis of a wide range of psychiatric disorders. Recently, numerous studies have associated the DNA methylation level of BDNF promoters with certain psychiatric phenotypes. In this review, we summarize data from current literature as well as from our own analysis with respect to the correlation of BDNF methylation changes with psychiatric disorders and address questions about whether DNA methylation related to the BDNF can be useful as biomarker for specific neuropsychiatric disorders.


Assuntos
Fator Neurotrófico Derivado do Encéfalo/genética , Metilação de DNA , Transtornos Mentais/genética , Animais , Biomarcadores/sangue , Encéfalo/metabolismo , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Bases de Dados Genéticas , Humanos , Fenótipo , Regiões Promotoras Genéticas
9.
Genome Med ; 7: 103, 2015 Oct 08.
Artigo em Inglês | MEDLINE | ID: mdl-26449484

RESUMO

BACKGROUND: The mechanisms by which genetic variants, such as single nucleotide polymorphisms (SNPs), identified in genome-wide association studies act to influence body mass remain unknown for most of these SNPs, which continue to puzzle the scientific community. Recent evidence points to the epigenetic and chromatin states of the genome as having important roles. METHODS: We genotyped 355 healthy young individuals for 52 known obesity-associated SNPs and obtained DNA methylation levels in their blood using the Illumina 450 K BeadChip. Associations between alleles and methylation at proximal cytosine residues were tested using a linear model adjusted for age, sex, weight category, and a proxy for blood cell type counts. For replication in other tissues, we used two open-access datasets (skin fibroblasts, n = 62; four brain regions, n = 121-133) and an additional dataset in subcutaneous and visceral fat (n = 149). RESULTS: We found that alleles at 28 of these obesity-associated SNPs associate with methylation levels at 107 proximal CpG sites. Out of 107 CpG sites, 38 are located in gene promoters, including genes strongly implicated in obesity (MIR148A, BDNF, PTPMT1, NR1H3, MGAT1, SCGB3A1, HOXC12, PMAIP1, PSIP1, RPS10-NUDT3, RPS10, SKOR1, MAP2K5, SIX5, AGRN, IMMP1L, ELP4, ITIH4, SEMA3G, POMC, ADCY3, SSPN, LGR4, TUFM, MIR4721, SULT1A1, SULT1A2, APOBR, CLN3, SPNS1, SH2B1, ATXN2L, and IL27). Interestingly, the associated SNPs are in known eQTLs for some of these genes. We also found that the 107 CpGs are enriched in enhancers in peripheral blood mononuclear cells. Finally, our results indicate that some of these associations are not blood-specific as we successfully replicated four associations in skin fibroblasts. CONCLUSIONS: Our results strongly suggest that many obesity-associated SNPs are associated with proximal gene regulation, which was reflected by association of obesity risk allele genotypes with differential DNA methylation. This study highlights the importance of DNA methylation and other chromatin marks as a way to understand the molecular basis of genetic variants associated with human diseases and traits.


Assuntos
Metilação de DNA , Obesidade/genética , Tecido Adiposo/metabolismo , Adolescente , Adulto , Idoso , Alelos , Encéfalo/metabolismo , Ilhas de CpG , Elementos Facilitadores Genéticos , Feminino , Fibroblastos/metabolismo , Humanos , Masculino , Pessoa de Meia-Idade , Obesidade/sangue , Polimorfismo de Nucleotídeo Único , Regiões Promotoras Genéticas , Adulto Jovem
10.
PLoS One ; 10(3): e0121964, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25821969

RESUMO

Spinal muscular atrophy (SMA) is a monogenic neurodegenerative disorder subdivided into four different types. Whole genome methylation analysis revealed 40 CpG sites associated with genes that are significantly differentially methylated between SMA patients and healthy individuals of the same age. To investigate the contribution of methylation changes to SMA severity, we compared the methylation level of found CpG sites, designed as "targets", as well as the nearest CpG sites in regulatory regions of ARHGAP22, CDK2AP1, CHML, NCOR2, SLC23A2 and RPL9 in three groups of SMA patients. Of notable interest, compared to type I SMA male patients, the methylation level of a target CpG site and one nearby CpG site belonging to the 5'UTR of SLC23A2 were significantly hypomethylated 19-22% in type III-IV patients. In contrast to type I SMA male patients, type III-IV patients demonstrated a 16% decrease in the methylation levels of a target CpG site, belonging to the 5'UTR of NCOR2. To conclude, this study validates the data of our previous study and confirms significant methylation changes in the SLC23A2 and NCOR2 regulatory regions correlates with SMA severity.


Assuntos
Metilação de DNA/genética , Correpressor 2 de Receptor Nuclear/genética , Transportadores de Sódio Acoplados à Vitamina C/genética , Atrofias Musculares Espinais da Infância/genética , Regiões 5' não Traduzidas/genética , Adolescente , Sequência de Bases , Estudos de Casos e Controles , Pré-Escolar , Ilhas de CpG/genética , Feminino , Humanos , Lactente , Masculino , Dados de Sequência Molecular
11.
PLoS Genet ; 10(9): e1004499, 2014 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-25187989

RESUMO

In all animals managing the size of individual meals and frequency of feeding is crucial for metabolic homeostasis. In the current study we demonstrate that the noradrenalin analogue octopamine and the cholecystokinin (CCK) homologue Drosulfakinin (Dsk) function downstream of TfAP-2 and Tiwaz (Twz) to control the number of meals in adult flies. Loss of TfAP-2 or Twz in octopaminergic neurons increased the size of individual meals, while overexpression of TfAP-2 significantly decreased meal size and increased feeding frequency. Of note, our study reveals that TfAP-2 and Twz regulate octopamine signaling to initiate feeding; then octopamine, in a negative feedback loop, induces expression of Dsk to inhibit consummatory behavior. Intriguingly, we found that the mouse TfAP-2 and Twz homologues, AP-2ß and Kctd15, co-localize in areas of the brain known to regulate feeding behavior and reward, and a proximity ligation assay (PLA) demonstrated that AP-2ß and Kctd15 interact directly in a mouse hypothalamus-derived cell line. Finally, we show that in this mouse hypothalamic cell line AP-2ß and Kctd15 directly interact with Ube2i, a mouse sumoylation enzyme, and that AP-2ß may itself be sumoylated. Our study reveals how two obesity-linked homologues regulate metabolic homeostasis by modulating consummatory behavior.


Assuntos
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Drosophila melanogaster/fisiologia , Comportamento Alimentar/fisiologia , Refeições/fisiologia , Obesidade/metabolismo , Obesidade/fisiopatologia , Animais , Linhagem Celular , Retroalimentação , Homeostase/fisiologia , Hipotálamo/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neurônios/metabolismo , Octopamina/metabolismo , Canais de Potássio/metabolismo , Fator de Transcrição AP-2/metabolismo
12.
Eur J Hum Genet ; 21(9): 988-93, 2013 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-23299920

RESUMO

Spinal muscular atrophy (SMA) is a monogenic disorder that is subdivided into four different types and caused by survival motor neuron gene 1 (SMN1) deletion. Discordant cases of SMA suggest that there exist additional severity modifying factors, apart from the SMN2 gene copy number. Here we performed the first genome-wide methylation profiling of SMA patients and healthy individuals to study the association of DNA methylation status with the severity of the SMA phenotype. We identified strong significant differences in methylation level between SMA patients and healthy controls in CpG sites close to the genes CHML, ARHGAP22, CYTSB, CDK2AP1 and SLC23A2. Interestingly, the CHML and ARHGAP22 genes are associated with the activity of Rab and Rho GTPases, which are important regulators of vesicle formation, actin dynamics, axonogenesis, processes that could be critical for SMA development. We suggest that epigenetic modifications may influence the severity of SMA and that these novel genetic positions could prove to be valuable biomarkers for the understanding of SMA pathogenesis.


Assuntos
Epigênese Genética , Atrofia Muscular Espinal/genética , Proteínas rab de Ligação ao GTP/genética , Proteínas rho de Ligação ao GTP/genética , Adolescente , Adulto , Estudos de Casos e Controles , Criança , Pré-Escolar , Ilhas de CpG , Metilação de DNA , Ontologia Genética , Predisposição Genética para Doença , Estudo de Associação Genômica Ampla , Humanos , Lactente , Masculino , Atrofia Muscular Espinal/patologia , Análise de Sequência de DNA , Adulto Jovem , Proteínas rab de Ligação ao GTP/metabolismo , Proteínas rho de Ligação ao GTP/metabolismo
13.
BMC Med Genet ; 12: 96, 2011 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-21762474

RESUMO

BACKGROUND: Spinal muscular atrophy (SMA type I, II and III) is an autosomal recessive neuromuscular disorder caused by mutations in the survival motor neuron gene (SMN1). SMN2 is a centromeric copy gene that has been characterized as a major modifier of SMA severity. SMA type I patients have one or two SMN2 copies while most SMA type II patients carry three SMN2 copies and SMA III patients have three or four SMN2 copies. The SMN1 gene produces a full-length transcript (FL-SMN) while SMN2 is only able to produce a small portion of the FL-SMN because of a splice mutation which results in the production of abnormal SMNΔ7 mRNA. METHODS: In this study we performed quantification of the SMN2 gene copy number in Russian patients affected by SMA type II and III (42 and 19 patients, respectively) by means of real-time PCR. Moreover, we present two families consisting of asymptomatic carriers of a homozygous absence of the SMN1 gene. We also developed a novel RT-qPCR-based assay to determine the FL-SMN/SMNΔ7 mRNA ratio as SMA biomarker. RESULTS: Comparison of the SMN2 copy number and clinical features revealed a significant correlation between mild clinical phenotype (SMA type III) and presence of four copies of the SMN2 gene. In both asymptomatic cases we found an increased number of SMN2 copies in the healthy carriers and a biallelic SMN1 absence. Furthermore, the novel assay revealed a difference between SMA patients and healthy controls. CONCLUSIONS: We suggest that the SMN2 gene copy quantification in SMA patients could be used as a prognostic tool for discrimination between the SMA type II and SMA type III diagnoses, whereas the FL-SMN/SMNΔ7 mRNA ratio could be a useful biomarker for detecting changes during SMA pharmacotherapy.


Assuntos
Dosagem de Genes , Atrofias Musculares Espinais da Infância/genética , Sequência de Bases , Estudos de Casos e Controles , Primers do DNA/genética , Éxons , Feminino , Expressão Gênica , Heterozigoto , Homozigoto , Humanos , Masculino , Linhagem , Fenótipo , Prognóstico , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Federação Russa , Deleção de Sequência , Proteína 1 de Sobrevivência do Neurônio Motor/genética , Proteína 2 de Sobrevivência do Neurônio Motor/genética
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