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1.
BMC Bioinformatics ; 22(1): 476, 2021 Oct 03.
Artigo em Inglês | MEDLINE | ID: mdl-34602053

RESUMO

BACKGROUND: Quantitative, reverse transcription PCR (qRT-PCR) is currently the gold-standard for SARS-CoV-2 detection and it is also used for detection of other virus. Manual data analysis of a small number of qRT-PCR plates per day is a relatively simple task, but automated, integrative strategies are needed if a laboratory is dealing with hundreds of plates per day, as is being the case in the COVID-19 pandemic. RESULTS: Here we present shinyCurves, an online shiny-based, free software to analyze qRT-PCR amplification data from multi-plate and multi-platform formats. Our shiny application does not require any programming experience and is able to call samples Positive, Negative or Undetermined for viral infection according to a number of user-defined settings, apart from providing a complete set of melting and amplification curve plots for the visual inspection of results. CONCLUSIONS: shinyCurves is a flexible, integrative and user-friendly software that speeds-up the analysis of massive qRT-PCR data from different sources, with the possibility of automatically producing and evaluating melting and amplification curve plots.


Assuntos
COVID-19 , Análise de Dados , Humanos , Pandemias , Reação em Cadeia da Polimerase em Tempo Real , SARS-CoV-2
2.
Nat Commun ; 12(1): 5095, 2021 08 24.
Artigo em Inglês | MEDLINE | ID: mdl-34429407

RESUMO

Maternal smoking during pregnancy (MSDP) contributes to poor birth outcomes, in part through disrupted placental functions, which may be reflected in the placental epigenome. Here we present a meta-analysis of the associations between MSDP and placental DNA methylation (DNAm) and between DNAm and birth outcomes within the Pregnancy And Childhood Epigenetics (PACE) consortium (N = 1700, 344 with MSDP). We identify 443 CpGs that are associated with MSDP, of which 142 associated with birth outcomes, 40 associated with gene expression, and 13 CpGs are associated with all three. Only two CpGs have consistent associations from a prior meta-analysis of cord blood DNAm, demonstrating substantial tissue-specific responses to MSDP. The placental MSDP-associated CpGs are enriched for environmental response genes, growth-factor signaling, and inflammation, which play important roles in placental function. We demonstrate links between placental DNAm, MSDP and poor birth outcomes, which may better inform the mechanisms through which MSDP impacts placental function and fetal growth.


Assuntos
Metilação de DNA , Desenvolvimento Fetal/efeitos dos fármacos , Desenvolvimento Fetal/genética , Placenta/metabolismo , Fumar/efeitos adversos , Epigênese Genética , Feminino , Heterogeneidade Genética , Humanos , Motivos de Nucleotídeos , Gravidez , Tabaco
3.
Environ Sci Technol ; 2021 Jul 27.
Artigo em Inglês | MEDLINE | ID: mdl-34314170

RESUMO

Results of studies on perfluoroalkyl substances (PFASs) and thyroid hormones (THs) are heterogeneous, and the mechanisms underlying the action of PFASs to target THs have not been fully characterized. We examined the relation between first-trimester maternal PFAS and TH levels and the role played by polymorphisms in the iodothyronine deiodinase 1 (DIO1) and 2 (DIO2) genes in this association. Our sample comprised 919 pregnant Spanish women (recruitment = 2003-2008) with measurements of perfluorohexanesulfonic acid (PFHxS), perfluorooctanoic acid (PFOA), perfluorooctanesulfonic acid (PFOS), perfluorononanoic acid (PFNA), thyroid-stimulating hormone (TSH), total triiodothyronine (TT3), and free thyroxine (FT4), and we genotyped for single-nucleotide polymorphisms in the DIO1 (rs2235544) and DIO2 (rs12885300) genes. We performed multivariate regression analyses between PFASs and THs and included the interaction term PFAS-genotypes in the models. PFHxS was associated with an increase in TSH (% change in outcome [95% CI] per 2-fold PFAS increase = 6.09 [-0.71, 13.4]), and PFOA and PFNA were associated with a decrease in TT3 (-7.17 [-13.5, -0.39] and -6.28 [-12.3, 0.12], respectively). We found stronger associations between PFOA, PFNA, and TT3 for DIO1-CC and DIO2-CT genotypes, although interaction p-values were not significant. In conclusion, this study found evidence of an inverse association between PFOA and TT3 levels. No clear effect modification by DIO enzyme genes was observed.

4.
Int J Mol Sci ; 22(11)2021 Jun 04.
Artigo em Inglês | MEDLINE | ID: mdl-34199930

RESUMO

Endometriosis is a common gynecological disorder that has been associated with endometrial, breast and epithelial ovarian cancers in epidemiological studies. Since complex diseases are a result of multiple environmental and genetic factors, we hypothesized that the biological mechanism underlying their comorbidity might be explained, at least in part, by shared genetics. To assess their potential genetic relationship, we performed a two-sample mendelian randomization (2SMR) analysis on results from public genome-wide association studies (GWAS). This analysis confirmed previously reported genetic pleiotropy between endometriosis and endometrial cancer. We present robust evidence supporting a causal genetic association between endometriosis and ovarian cancer, particularly with the clear cell and endometrioid subtypes. Our study also identified genetic variants that could explain those associations, opening the door to further functional experiments. Overall, this work demonstrates the value of genomic analyses to support epidemiological data, and to identify targets of relevance in multiple disorders.


Assuntos
Neoplasias do Endométrio/epidemiologia , Endometriose/epidemiologia , Endométrio/patologia , Predisposição Genética para Doença , Neoplasias Hormônio-Dependentes/epidemiologia , Neoplasias Ovarianas/epidemiologia , Polimorfismo de Nucleotídeo Único , Neoplasias do Endométrio/genética , Neoplasias do Endométrio/patologia , Endometriose/genética , Endometriose/patologia , Feminino , Estudo de Associação Genômica Ampla , Humanos , Neoplasias Hormônio-Dependentes/genética , Neoplasias Hormônio-Dependentes/patologia , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/patologia , Fatores de Risco , Espanha/epidemiologia
5.
Int Rev Cell Mol Biol ; 358: 1-45, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33707051

RESUMO

Celiac Disease (CeD) is an immune-mediated complex disease that is triggered by the ingestion of gluten and develops in genetically susceptible individuals. It has been known for a long time that the Human Leucocyte Antigen (HLA) molecules DQ2 and DQ8 are necessary, although not sufficient, for the disease development, and therefore other susceptibility genes and (epi)genetic events must participate in CeD pathogenesis. The advances in Genomics during the last 15 years have made CeD one of the immune-related disorders with the best-characterized genetic component. In the present work, we will first review the main Genome-Wide Association Studies (GWAS) carried out in the disorder, and emphasize post-GWAS discoveries, including diverse integrative strategies, SNP prioritization approaches, and insights into the Microbiome through the host Genomics. Second, we will explore CeD-related Epigenetics and Epigenomics, mostly focusing on the emerging knowledge of the celiac methylome, and the vast but yet under-explored non-coding RNA (ncRNA) landscape. We conclude that much has been done in the field although there are still completely unvisited areas in the post-Genomics of CeD. Chromatin conformation and accessibility, and Epitranscriptomics are promising domains that need to be unveiled to complete the big picture of the celiac Genome.

6.
Sci Rep ; 11(1): 313, 2021 01 11.
Artigo em Inglês | MEDLINE | ID: mdl-33432064

RESUMO

Although genome-wide association studies have identified single nucleotide polymorphisms (SNPs) associated with the susceptibility to Mycobacterium avium subsp. paratuberculosis (MAP) infection, only a few functional mutations for bovine paratuberculosis (PTB) have been characterized. Expression quantitative trait loci (eQTLs) are genetic variants typically located in gene regulatory regions that alter gene expression in an allele-specific manner. eQTLs can be considered as functional links between genomic variants, gene expression, and ultimately phenotype. In the current study, peripheral blood (PB) and ileocecal valve (ICV) gene expression was quantified by RNA-Seq from fourteen Holstein cattle with no lesions and with PTB-associated histopathological lesions in gut tissues. Genotypes were generated from the Illumina LD EuroG10K BeadChip. The associations between gene expression levels (normalized read counts) and genetic variants were analyzed by a linear regression analysis using R Matrix eQTL 2.2. This approach allowed the identification of 192 and 48 cis-eQTLs associated with the expression of 145 and 43 genes in the PB and ICV samples, respectively. To investigate potential relationships between these cis-eQTLs and MAP infection, a case-control study was performed using the genotypes for all the identified cis-eQTLs and phenotypical data (histopathology, ELISA for MAP-antibodies detection, tissue PCR, and bacteriological culture) of 986 culled cows. Our results suggested that the heterozygous genotype in the cis-eQTL-rs43744169 (T/C) was associated with the up-regulation of the MDS1 and EVI1 complex (MECOM) expression, with positive ELISA, PCR, and bacteriological culture results, and with increased risk of progression to clinical PTB. As supporting evidence, the presence of the minor allele was associated with higher MECOM levels in plasma samples from infected cows and with increased MAP survival in an ex-vivo macrophage killing assay. Moreover, the presence of the two minor alleles in the cis-eQTL-rs110345285 (C/C) was associated with the dysregulation of the eukaryotic elongation factor 1-α2 (eEF1A2) expression and with increased ELISA (OD) values. Finally, the presence of the minor allele in the cis-eQTL rs109859270 (C/T) was associated with the up-regulation of the U1 spliceosomal RNA expression and with an increased risk of progression to clinical PTB. The introduction of these novel functional variants into marker-assisted breeding programs is expected to have a relevant effect on PTB control.


Assuntos
Regulação da Expressão Gênica , Proteína do Locus do Complexo MDS1 e EVI1/genética , Paratuberculose/genética , Fator 1 de Elongação de Peptídeos/genética , Locos de Características Quantitativas/genética , Spliceossomos/genética , Animais , Bovinos , Predisposição Genética para Doença/genética , Polimorfismo de Nucleotídeo Único
7.
Nutrients ; 12(5)2020 May 14.
Artigo em Inglês | MEDLINE | ID: mdl-32423041

RESUMO

Celiac disease (CeD) is a complex immune-mediated inflammatory condition triggered by the ingestion of gluten in genetically predisposed individuals. Literature suggests that alterations in gut microbiota composition and function precede the onset of CeD. Considering that microbiota is partly determined by host genetics, we speculated that the genetic makeup of CeD patients could elicit disease development through alterations in the intestinal microbiota. To evaluate potential causal relationships between gut microbiota and CeD, we performed a two-sample Mendelian randomization analysis (2SMR). Exposure data were obtained from the raw results of a previous genome-wide association study (GWAS) of gut microbiota and outcome data from summary statistics of CeD GWAS and Immunochip studies. We identified a number of putative associations between gut microbiota single nucleotide polymorphisms (SNPs) associated with CeD. Regarding bacterial composition, most of the associated SNPs were related to Firmicutes phylum, whose relative abundance has been previously reported to be altered in CeD patients. In terms of functional units, we linked a number of SNPs to several bacterial metabolic pathways that seemed to be related to CeD. Overall, this study represented the first 2SMR approach to elucidate the relationship between microbiome and CeD.


Assuntos
Doença Celíaca/genética , Doença Celíaca/microbiologia , Microbioma Gastrointestinal/genética , Predisposição Genética para Doença/epidemiologia , Predisposição Genética para Doença/genética , Estudo de Associação Genômica Ampla , Humanos , Análise da Randomização Mendeliana , Polimorfismo de Nucleotídeo Único/genética
8.
Epigenetics ; 15(10): 1068-1082, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32281463

RESUMO

Abnormal DNA methylation has been described in human inflammatory conditions of the gastrointestinal tract, such as inflammatory bowel disease (IBD). As other complex diseases, IBD results from the balance between genetic predisposition and environmental exposures. As such, DNA methylation may be the consequence (and potential effector) of both, genetic susceptibility variants and/or environmental signals such as cytokine exposure. We attempted to discern between these two non-excluding possibilities by performing a combined analysis of published DNA methylation data in intestinal mucosal cells of IBD and control samples. We identified abnormal DNA methylation at different levels: deviation from mean methylation signals at site and region levels, and differential variability. A fraction of such changes is associated with genetic polymorphisms linked to IBD susceptibility. In addition, by comparing with another intestinal inflammatory condition (i.e., coeliac disease) we propose that aberrant DNA methylation can also be the result of unspecific processes such as chronic inflammation. Our characterization suggests that IBD methylomes combine intrinsic and extrinsic responses in intestinal mucosal cells, and could point to knowledge-based biomarkers of IBD detection and progression.


Assuntos
Epigenoma , Doenças Inflamatórias Intestinais/genética , Mucosa Intestinal/metabolismo , Adolescente , Adulto , Idoso , Criança , Metilação de DNA , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Locos de Características Quantitativas
9.
Exp Dermatol ; 29(1): 39-50, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31602702

RESUMO

Tumor cell invasion is one of the key processes during cancer progression, leading to life-threatening metastatic lesions in melanoma. As methylation of cancer-related genes plays a fundamental role during tumorigenesis and may lead to cellular plasticity which promotes invasion, our aim was to identify novel epigenetic markers on selected invasive melanoma cells. Using Illumina BeadChip assays and Affymetrix Human Gene 1.0 microarrays, we explored the DNA methylation landscape of selected invasive melanoma cells and examined the impact of DNA methylation on gene expression patterns. Our data revealed predominantly hypermethylated genes in the invasive cells affecting the neural crest differentiation pathway and regulation of the actin cytoskeleton. Integrative analysis of the methylation and gene expression profiles resulted in a cohort of hypermethylated genes (IL12RB2, LYPD6B, CHL1, SLC9A3, BAALC, FAM213A, SORCS1, GPR158, FBN1 and ADORA2B) with decreased expression. On the other hand, hypermethylation in the gene body of the EGFR and RBP4 genes was positively correlated with overexpression of the genes. We identified several methylation changes that can have role during melanoma progression, including hypermethylation of the promoter regions of the ARHGAP22 and NAV2 genes that are commonly altered in locally invasive primary melanomas as well as during metastasis. Interestingly, the down-regulation of the methylcytosine dioxygenase TET2 gene, which regulates DNA methylation, was associated with hypermethylated promoter region of the gene. This can probably lead to the observed global hypermethylation pattern of invasive cells and might be one of the key changes during the development of malignant melanoma cells.


Assuntos
Metilação de DNA , Melanoma/genética , Melanoma/secundário , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/patologia , Citoesqueleto de Actina/genética , Citoesqueleto de Actina/metabolismo , Diferenciação Celular/genética , Linhagem Celular Tumoral , DNA Helicases/genética , Proteínas de Ligação a DNA/genética , Epigênese Genética , Proteínas Ativadoras de GTPase/genética , Expressão Gênica/genética , Perfilação da Expressão Gênica , Humanos , Invasividade Neoplásica/genética , Metástase Neoplásica/genética , Fenótipo , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas/genética
10.
Nucleic Acids Res ; 47(19): 10072-10085, 2019 11 04.
Artigo em Inglês | MEDLINE | ID: mdl-31665742

RESUMO

Mitochondrial dysfunction plays critical roles in cancer development and related therapeutic response; however, exact molecular mechanisms remain unclear. Recently, alongside the discovery of mitochondrial-specific DNA methyltransferases, global and site-specific methylation of the mitochondrial genome has been described. Investigation of any functional consequences however remains unclear and debated due to insufficient evidence of the quantitative degree and frequency of mitochondrial DNA (mtDNA) methylation. This study uses WGBS to provide the first quantitative report of mtDNA methylation at single base pair resolution. The data show that mitochondrial genomes are extensively methylated predominantly at non-CpG sites. Importantly, these methylation patterns display notable differences between normal and cancer cells. Furthermore, knockdown of DNA methyltransferase enzymes resulted in a marked global reduction of mtDNA methylation levels, indicating these enzymes may be associated with the establishment and/or maintenance of mtDNA methylation. DNMT3B knockdown cells displayed a comparatively pronounced global reduction in mtDNA methylation with concomitant increases in gene expression, suggesting a potential functional link between methylation and gene expression. Together these results demonstrate reproducible, non-random methylation patterns of mtDNA and challenge the notion that mtDNA is lowly methylated. This study discusses key differences in methodology that suggest future investigations must allow for techniques that assess both CpG and non-CpG methylation.


Assuntos
DNA (Citosina-5-)-Metiltransferases/genética , Metilação de DNA/genética , DNA Mitocondrial/genética , Regulação da Expressão Gênica/genética , Animais , Ilhas de CpG/genética , Humanos , Mitocôndrias/genética
11.
Epigenetics ; 14(12): 1177-1182, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31250700

RESUMO

Illumina HumanMethylation450 BeadChip (450K) has been commonly used to investigate DNA methylation in human tissues. Recently, it has been replaced by Illumina HumanMethylationEPIC BeadChip (EPIC) covering over 850,000 CpGs distributed genome-wide. Many consortia have now datasets coming from both arrays and aspire to analyze the two together. The placenta shows a high number of intermediate methylation levels and is often investigated for obstetric/birth outcomes, and potentially for long-term programming in offspring. We performed a systematic comparison between the two arrays using 108 duplicate placental samples from Gen3G birth cohort. We find that placenta shows a high per-sample correlation between the arrays, and higher median correlations at individual CpGs than those reported for blood. We identify 26,340 probes with absolute difference in per cent methylation >10%. We conclude that EPIC and 450K placental data can be combined, and we provide two lists of CpGs that should be excluded to avoid misleading results.


Assuntos
Metilação de DNA , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Placenta/metabolismo , Análise de Sequência de DNA/métodos , Adulto , Ilhas de CpG , Feminino , Sequenciamento de Nucleotídeos em Larga Escala/normas , Humanos , Análise de Sequência com Séries de Oligonucleotídeos/normas , Gravidez , Reprodutibilidade dos Testes , Análise de Sequência de DNA/normas
12.
Nucleic Acids Res ; 47(14): e81, 2019 08 22.
Artigo em Inglês | MEDLINE | ID: mdl-31049595

RESUMO

Bisulfite amplicon sequencing has become the primary choice for single-base methylation quantification of multiple targets in parallel. The main limitation of this technology is a preferential amplification of an allele and strand in the PCR due to methylation state. This effect, known as 'PCR bias', causes inaccurate estimation of the methylation levels and calibration methods based on standard controls have been proposed to correct for it. Here, we present a Bayesian calibration tool, MethylCal, which can analyse jointly all CpGs within a CpG island (CGI) or a Differentially Methylated Region (DMR), avoiding 'one-at-a-time' CpG calibration. This enables more precise modeling of the methylation levels observed in the standard controls. It also provides accurate predictions of the methylation levels not considered in the controlled experiment, a feature that is paramount in the derivation of the corrected methylation degree. We tested the proposed method on eight independent assays (two CpG islands and six imprinting DMRs) and demonstrated its benefits, including the ability to detect outliers. We also evaluated MethylCal's calibration in two practical cases, a clinical diagnostic test on 18 patients potentially affected by Beckwith-Wiedemann syndrome, and 17 individuals with celiac disease. The calibration of the methylation levels obtained by MethylCal allows a clearer identification of patients undergoing loss or gain of methylation in borderline cases and could influence further clinical or treatment decisions.


Assuntos
Teorema de Bayes , Biologia Computacional/métodos , Ilhas de CpG/genética , Metilação de DNA , Impressão Genômica , Análise de Sequência de DNA/métodos , Algoritmos , Síndrome de Beckwith-Wiedemann/diagnóstico , Síndrome de Beckwith-Wiedemann/genética , Síndrome de Beckwith-Wiedemann/terapia , Calibragem , Doença Celíaca/diagnóstico , Doença Celíaca/genética , Doença Celíaca/terapia , Humanos , Canais de Potássio de Abertura Dependente da Tensão da Membrana/genética , RNA Longo não Codificante/genética , Reprodutibilidade dos Testes , Sensibilidade e Especificidade
13.
Hum Mol Genet ; 28(18): 3037-3042, 2019 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-31127932

RESUMO

Celiac disease (CeD) is an immune-mediated enteropathy with a strong genetic component where the main environmental trigger is dietary gluten, and currently a correct diagnosis of the disease is impossible if gluten-free diet (GFD) has already been started. We hypothesized that merging different levels of genomic information through Mendelian randomization (MR) could help discover genetic biomarkers useful for CeD diagnosis. MR was performed using public databases of expression quantitative trait loci (QTL) and methylation QTL as exposures and the largest CeD genome-wide association study conducted to date as the outcome, in order to identify potential causal genes. As a result, we identified UBE2L3, an ubiquitin ligase located in a CeD-associated region. We interrogated the expression of UBE2L3 in an independent data set of peripheral blood mononuclear cells (PBMCs) and found that its expression is altered in CeD patients on GFD when compared to non-celiac controls. The relative expression of UBE2L3 isoforms predicts CeD with 100% specificity and sensitivity and could be used as a diagnostic marker, especially in the absence of gluten consumption. This approach could be applicable to other diseases where diagnosis of asymptomatic patients can be complicated.


Assuntos
Doença Celíaca/sangue , Doença Celíaca/genética , Predisposição Genética para Doença , Estudo de Associação Genômica Ampla , Análise da Randomização Mendeliana , Transcriptoma , Biomarcadores , Doença Celíaca/diagnóstico , Doença Celíaca/dietoterapia , Diagnóstico Diferencial , Dieta Livre de Glúten , Humanos , Polimorfismo de Nucleotídeo Único , Prognóstico , Locos de Características Quantitativas , Característica Quantitativa Herdável , Curva ROC
14.
Sci Rep ; 9(1): 1298, 2019 02 04.
Artigo em Inglês | MEDLINE | ID: mdl-30718669

RESUMO

The Human Leucocyte Antigen (HLA) locus and other DNA sequence variants identified in Genome-Wide Association (GWA) studies explain around 50% of the heritability of celiac disease (CD). However, the pathogenesis of CD could be driven by other layers of genomic information independent from sequence variation, such as DNA methylation, and it is possible that allele-specific methylation explains part of the SNP associations. Since the DNA methylation landscape is expected to be different among cell types, we analyzed the methylome of the epithelial and immune cell populations of duodenal biopsies in CD patients and controls separately. We found a cell type-specific methylation signature that includes genes mapping to the HLA region, namely TAP1 and HLA-B. We also performed Immunochip SNP genotyping of the same samples and interrogated the expression of some of the affected genes. Our analysis revealed that the epithelial methylome is characterized by the loss of CpG island (CGI) boundaries, often associated to altered gene expression, and by the increased variability of the methylation across the samples. The overlap between differentially methylated positions (DMPs) and CD-associated SNPs or variants contributing to methylation quantitative trait loci (mQTLs) is minimal. In contrast, there is a notable enrichment of mQTLs among the most significant CD-associated SNPs. Our results support the notion that DNA methylation alterations constitute a genotype-independent event and confirm its role in the HLA region (apart from the well-known, DQ allele-specific effect). Finally, we find that a fraction of the CD-associated variants could exert its phenotypic effect through DNA methylation.


Assuntos
Doença Celíaca/etiologia , Metilação de DNA , Epigenoma , Genótipo , Antígenos HLA/genética , Mucosa Intestinal/imunologia , Mucosa Intestinal/metabolismo , Biópsia , Doença Celíaca/metabolismo , Doença Celíaca/patologia , Ilhas de CpG , Feminino , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Estudo de Associação Genômica Ampla , Antígenos HLA/imunologia , Humanos , Mucosa Intestinal/patologia , Masculino , Regiões Promotoras Genéticas
15.
Genes (Basel) ; 9(5)2018 May 10.
Artigo em Inglês | MEDLINE | ID: mdl-29748492

RESUMO

The aim of this study was to construct celiac co-expression patterns at a whole genome level and to identify transcription factors (TFs) that could drive the gliadin-related changes in coordination of gene expression observed in celiac disease (CD). Differential co-expression modules were identified in the acute and chronic responses to gliadin using expression data from a previous microarray study in duodenal biopsies. Transcription factor binding site (TFBS) and Gene Ontology (GO) annotation enrichment analyses were performed in differentially co-expressed genes (DCGs) and selection of candidate regulators was performed. Expression of candidates was measured in clinical samples and the activation of the TFs was further characterized in C2BBe1 cells upon gliadin challenge. Enrichment analyses of the DCGs identified 10 TFs and five were selected for further investigation. Expression changes related to active CD were detected in four TFs, as well as in several of their in silico predicted targets. The activation of TFs was further characterized in C2BBe1 cells upon gliadin challenge, and an increase in nuclear translocation of CAMP Responsive Element Binding Protein 1 (CREB1) and IFN regulatory factor-1 (IRF1) in response to gliadin was observed. Using transcriptome-wide co-expression analyses we are able to propose novel genes involved in CD pathogenesis that respond upon gliadin stimulation, also in non-celiac models.

16.
J Pediatr Gastroenterol Nutr ; 67(2): 225-231, 2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-29601440

RESUMO

OBJECTIVE: The aim of the study is to identify additional celiac disease associated loci in the major histocompatibility complex (MHC) independent from classical HLA risk alleles (HLA-DR3-DQ2) and to characterize their potential functional impact in celiac disease pathogenesis at the intestinal level. METHODS: We performed a high-resolution single-nucleotide polymorphism (SNP) genotyping of the MHC region, comparing HLA-DR3 homozygous celiac patients and non-celiac controls carrying a single copy of the B8-DR3-DQ2 conserved extended haplotype. Expression level of potential novel risk genes was determined by RT-PCR in intestinal biopsies and in intestinal and immune cells isolated from control and celiac individuals. Small interfering RNA-driven silencing of selected genes was performed in the intestinal cell line T84. RESULTS: MHC genotyping revealed 2 associated SNPs, one located in TRIM27 gene and another in the non-coding gene HCG14. After stratification analysis, only HCG14 showed significant association independent from HLA-DR-DQ loci. Expression of HCG14 was slightly downregulated in epithelial cells isolated from duodenal biopsies of celiac patients, and eQTL analysis revealed that polymorphisms in HCG14 region were associated with decreased NOD1 expression in duodenal intestinal cells. CONCLUSIONS: We have successfully employed a conserved extended haplotype-matching strategy and identified a novel additional celiac disease risk variant in the lncRNA HCG14. This lncRNA seems to regulate the expression of NOD1 in an allele-specific manner. Further functional studies are needed to clarify the role of HCG14 in the regulation of gene expression and to determine the molecular mechanisms by which the risk variant in HCG14 contributes to celiac disease pathogenesis.


Assuntos
Doença Celíaca/genética , Predisposição Genética para Doença , Antígeno HLA-DR3/genética , Proteína Adaptadora de Sinalização NOD1/metabolismo , RNA Longo não Codificante/genética , Estudos de Casos e Controles , Doença Celíaca/metabolismo , Doença Celíaca/patologia , Criança , Feminino , Humanos , Masculino , Reação em Cadeia da Polimerase , Polimorfismo de Nucleotídeo Único
17.
Int J Cancer ; 143(3): 597-609, 2018 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-29574700

RESUMO

The large geographic variations in the incidence of gastric cancer (GC) are likely due to differential environmental exposures, in particular to Helicobacter pylori (H. pylori) infection. We aimed to investigate the impact of H. pylori on the epigenome in normal gastric mucosa and methylation changes associated with cancer risk independent of H. pylori. A discovery set of normal gastric mucosa from GC cases (n = 42) and controls (n = 42), nested in a large case-control study and stratified by H. pylori status, were subjected to genome-wide methylation profiling. Single-nucleotide polymorphism arrays from peripheral blood leukocytes were used to conduct methylation quantitative trait loci (mQTL) analysis. A validation set of gastric mucosa samples (n = 180) was used in the replication phase. We found 1,924 differentially methylated positions (DMPs) and 438 differentially methylated regions (DMRs) associated with H. pylori infection, most of which were hypermethylated. Significant methylation alterations identified in the initial set were successfully replicated. Furthermore, the H. pylori-associated DMP/Rs showed marked stability ('epigenetic memory') after H. pylori clearance. Interestingly, we found 152 DMRs associated with cancer risk independent of the H. pylori status in normal gastric mucosa. The methylation score derived from three biomarkers was a strong predictor of GC. Finally, the mQTL analysis indicated that the H. pylori- and cancer-specific methylation signatures were minimally affected by genetic variation. The comprehensively characterized methylome changes associated with H. pylori infection and GC risk in our study might serve as potential biomarkers for early cancer progression in tumour-free gastric mucosa.


Assuntos
Metilação de DNA , Mucosa Gástrica/metabolismo , Infecções por Helicobacter/complicações , Neoplasias Gástricas/etiologia , Transcriptoma , Biomarcadores Tumorais , Biópsia , Estudos de Casos e Controles , Ilhas de CpG , Elementos Facilitadores Genéticos , Mucosa Gástrica/patologia , Perfilação da Expressão Gênica , Estudo de Associação Genômica Ampla , Humanos , Razão de Chances , Regiões Promotoras Genéticas , Locos de Características Quantitativas , Curva ROC , Neoplasias Gástricas/patologia
18.
Epigenetics ; 12(11): 964-972, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29099283

RESUMO

Breast cancer (BC) encompasses heterogeneous pathologies with different subtypes exhibiting distinct molecular changes, including those related to DNA methylation. However, the role of these changes in mediating BC heterogeneity is poorly understood. Lowly methylated regions (LMRs), non-CpG island loci that usually contain transcription factor (TF) binding sites, have been suggested to act as regulatory elements that define cellular identity. In this study, we aimed to identify the key subtype-specific TFs that may lead to LMR generation and shape the BC methylome and transcription program. We initially used whole-genome bisulfite sequencing (WGBS) data available at The Cancer Genome Atlas (TCGA) portal to identify subtype-specific LMRs. Differentially methylated regions (DMRs) within the BC PAM50 subtype-specific LMRs were selected by comparing tumors and normal tissues in a larger TCGA cohort assessed by HumanMethylation450 BeadChip (450K) arrays and TF enrichment analyses were performed. To assess the impact of LMRs on gene expression, TCGA RNA sequencing data were downloaded and Pearson correlations between methylation levels of loci presenting subtype-specific TF motifs and expression of the nearest genes were calculated. WGBS methylome data revealed a large number of LMRs for each of the BC subtypes. Analysis of these LMRs in the 450K datasets available for a larger sample set identified 7,765, 5,657, and 19 differentially methylated positions (DMPs) between normal adjacent tissues and tumor tissues from basal, luminal, and HER2-enriched subtypes, respectively. Unsupervised clustering showed that the discriminatory power of the top DMPs was remarkably strong for basal BC. Interestingly, in this particular subtype, we found 4,409 differentially hypomethylated positions grouped into 1,185 DMRs with a strong enrichment for the early B-cell factor 1 (EBF1) motifs. The methylation levels of the DMRs containing EBF1 motifs showed a strong negative correlation with the expression of 719 nearby genes, including BTS2 and CD74, two oncogenes known to be specific for basal BC subtype and for poor outcome. This study identifies LMRs specific to the three main BC subtypes and reveals EBF1 as a potentially important regulator of BC subtype-specific methylation and gene expression program.


Assuntos
Neoplasias da Mama/genética , Metilação de DNA , Epigênese Genética , Genes Modificadores , Transativadores/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos
19.
Genome Med ; 9(1): 33, 2017 04 05.
Artigo em Inglês | MEDLINE | ID: mdl-28381277

RESUMO

BACKGROUND: Head and neck squamous cell carcinomas (HNSCCs) represent a heterogeneous group of cancers for which human papilloma virus (HPV) infection is an emerging risk factor. Previous studies showed promoter hypermethylation in HPV(+) oropharyngeal cancers, but only few consistent target genes have been so far described, and the evidence of a functional impact on gene expression is still limited. METHODS: We performed global and stratified pooled analyses of epigenome-wide data in HNSCCs based on the Illumina HumanMethylation450 bead-array data in order to identify tissue-specific components and common viral epigenetic targets in HPV-associated tumours. RESULTS: We identified novel differentially methylated CpGs and regions associated with viral infection that are independent of the anatomic site. In particular, most hypomethylated regions were characterized by a marked loss of CpG island boundaries, which showed significant correlations with expression of neighbouring genes. Moreover, a subset of only five CpGs in a few hypomethylated regions predicted HPV status with a high level of specificity in different cohorts. Finally, this signature was a better predictor of survival compared with HPV status determined by viral gene expression by RNA sequencing in The Cancer Genome Atlas cohort. CONCLUSIONS: We identified a novel epigenetic signature of HPV infection in HNSCCs which is independent of the anatomic site, is functionally correlated with gene expression and may be leveraged for improved stratification of prognosis in HNSCCs.


Assuntos
Carcinoma de Células Escamosas/genética , Metilação de DNA , Neoplasias de Cabeça e Pescoço/genética , Infecções por Papillomavirus/complicações , Regiões Promotoras Genéticas , Adulto , Idoso , Idoso de 80 Anos ou mais , Carcinoma de Células Escamosas/etiologia , Carcinoma de Células Escamosas/metabolismo , Ilhas de CpG , Feminino , Regulação da Expressão Gênica , Genoma Humano , Neoplasias de Cabeça e Pescoço/etiologia , Neoplasias de Cabeça e Pescoço/metabolismo , Humanos , Masculino , Pessoa de Meia-Idade , Carcinoma de Células Escamosas de Cabeça e Pescoço , Adulto Jovem
20.
Science ; 352(6281): 91-5, 2016 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-27034373

RESUMO

Recent studies have implicated long noncoding RNAs (lncRNAs) as regulators of many important biological processes. Here we report on the identification and characterization of a lncRNA, lnc13, that harbors a celiac disease-associated haplotype block and represses expression of certain inflammatory genes under homeostatic conditions. Lnc13 regulates gene expression by binding to hnRNPD, a member of a family of ubiquitously expressed heterogeneous nuclear ribonucleoproteins (hnRNPs). Upon stimulation, lnc13 levels are reduced, thereby allowing increased expression of the repressed genes. Lnc13 levels are significantly decreased in small intestinal biopsy samples from patients with celiac disease, which suggests that down-regulation of lnc13 may contribute to the inflammation seen in this disease. Furthermore, the lnc13 disease-associated variant binds hnRNPD less efficiently than its wild-type counterpart, thus helping to explain how these single-nucleotide polymorphisms contribute to celiac disease.


Assuntos
Doença Celíaca/genética , Predisposição Genética para Doença , Inflamação/genética , RNA Longo não Codificante/genética , Animais , Sequência de Bases , Doença Celíaca/patologia , Regulação para Baixo , Regulação da Expressão Gênica , Haplótipos , Ribonucleoproteínas Nucleares Heterogêneas/genética , Humanos , Camundongos , Dados de Sequência Molecular , Polimorfismo de Nucleotídeo Único
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