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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.
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
3.
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.

4.
Gut ; 2021 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-33526437

RESUMO

OBJECTIVES: Coeliac disease (CD) is a complex autoimmune disorder that develops in genetically susceptible individuals. Dietary gluten triggers an immune response for which the only available treatment so far is a strict, lifelong gluten free diet. Human leucocyte antigen (HLA) genes and several non-HLA regions have been associated with the genetic susceptibility to CD, but their role in the pathogenesis of the disease is still essentially unknown, making it complicated to develop much needed non-dietary treatments. Here, we describe the functional involvement of a CD-associated single-nucleotide polymorphism (SNP) located in the 5'UTR of XPO1 in the inflammatory environment characteristic of the coeliac intestinal epithelium. DESIGN: The function of the CD-associated SNP was investigated using an intestinal cell line heterozygous for the SNP, N6-methyladenosine (m6A)-related knock-out and HLA-DQ2 mice, and human samples from patients with CD. RESULTS: Individuals harbouring the risk allele had higher m6A methylation in the 5'UTR of XPO1 RNA, rendering greater XPO1 protein amounts that led to downstream nuclear factor kappa B (NFkB) activity and subsequent inflammation. Furthermore, gluten exposure increased overall m6A methylation in humans as well as in in vitro and in vivo models. CONCLUSION: We identify a novel m6A-XPO1-NFkB pathway that is activated in CD patients. The findings will prompt the development of new therapeutic approaches directed at m6A proteins and XPO1, a target under evaluation for the treatment of intestinal disorders.

5.
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
6.
PLoS Genet ; 16(10): e1008718, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-33045005

RESUMO

The genetic background of childhood body mass index (BMI), and the extent to which the well-known associations of childhood BMI with adult diseases are explained by shared genetic factors, are largely unknown. We performed a genome-wide association study meta-analysis of BMI in 61,111 children aged between 2 and 10 years. Twenty-five independent loci reached genome-wide significance in the combined discovery and replication analyses. Two of these, located near NEDD4L and SLC45A3, have not previously been reported in relation to either childhood or adult BMI. Positive genetic correlations of childhood BMI with birth weight and adult BMI, waist-to-hip ratio, diastolic blood pressure and type 2 diabetes were detected (Rg ranging from 0.11 to 0.76, P-values <0.002). A negative genetic correlation of childhood BMI with age at menarche was observed. Our results suggest that the biological processes underlying childhood BMI largely, but not completely, overlap with those underlying adult BMI. The well-known observational associations of BMI in childhood with cardio-metabolic diseases in adulthood may reflect partial genetic overlap, but in light of previous evidence, it is also likely that they are explained through phenotypic continuity of BMI from childhood into adulthood.


Assuntos
Doenças Cardiovasculares/genética , Diabetes Mellitus Tipo 2/genética , Predisposição Genética para Doença , Proteínas de Transporte de Monossacarídeos/genética , Ubiquitina-Proteína Ligases Nedd4/genética , Adolescente , Adulto , Pressão Sanguínea , Índice de Massa Corporal , Fatores de Risco Cardiometabólico , Doenças Cardiovasculares/patologia , Criança , Pré-Escolar , Diabetes Mellitus Tipo 2/patologia , Feminino , Estudo de Associação Genômica Ampla/métodos , Humanos , Masculino , Menarca/genética , Análise da Randomização Mendeliana , Relação Cintura-Quadril
7.
ACS Appl Mater Interfaces ; 12(25): 27917-27929, 2020 Jun 24.
Artigo em Inglês | MEDLINE | ID: mdl-32464047

RESUMO

Local heat generation from magnetic nanoparticles (MNPs) exposed to alternating magnetic fields can revolutionize cancer treatment. However, the application of MNPs as anticancer agents is limited by serious drawbacks. Foremost among these are the fast uptake and biodegradation of MNPs by cells and the unpredictable magnetic behavior of the MNPs when they accumulate within or around cells and tissues. In fact, several studies have reported that the heating power of MNPs is severely reduced in the cellular environment, probably due to a combination of increased viscosity and strong NP agglomeration. Herein, we present an optimized protocol to coat magnetite (Fe3O4) NPs larger than 20 nm (FM-NPs) with high molecular weight PEG molecules that avoid collective coatings, prevent the formation of large clusters of NPs and keep constant their high heating performance in environments with very different ionic strengths and viscosities (distilled water, physiological solutions, agar and cell culture media). The great reproducibility and reliability of the heating capacity of this FM-NP@PEG system in such different environments has been confirmed by AC magnetometry and by more conventional calorimetric measurements. The explanation of this behavior has been shown to lie in preserving as much as possible the magnetic single domain-type behavior of nearly isolated NPs. In vitro endocytosis experiments in a colon cancer-derived cell line indicate that FM-NP@PEG formulations with PEGs of higher molecular weight (20 kDa) are more resistant to endocytosis than formulations with smaller PEGs (5 kDa), showing quite large uptake mean-life (τ > 5 h) in comparison with other NP systems. The in vitro magnetic hyperthermia was performed at 21 mT and 650 kHz during 1 h in a pre-endocytosis stage and complete cell death was achieved 48 h posthyperthermia. These optimal FM-NP@PEG formulations with high resistance to endocytosis and predictable magnetic response will aid the progress and accuracy of the emerging era of theranostics.


Assuntos
Ágar , Nanopartículas de Magnetita/química , Polietilenoglicóis/química , Água , Calorimetria , Linhagem Celular Tumoral , Endocitose/fisiologia , Humanos , Hipertermia Induzida/métodos , Magnetometria
8.
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
9.
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
10.
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
11.
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
12.
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
13.
Sci Rep ; 9(1): 4220, 2019 03 12.
Artigo em Inglês | MEDLINE | ID: mdl-30862814

RESUMO

N6-methyladenosine (m6A) is the most common and abundant RNA modification. Recent studies have shown its importance in the regulation of several biological processes, including the immune response, and different approaches have been developed in order to map and quantify m6A marks. However, site specific detection of m6A methylation has been technically challenging, and existing protocols are long and tedious and often involve next-generation sequencing. Here, we describe a simple RT-QPCR based approach for the relative quantification of candidate m6A regions that takes advantage of the diminished capacity of BstI enzyme to retrotranscribe m6A residues. Using this technique, we have been able to confirm the recently described m6A methylation in the 3'UTR of SOCS1 and SOCS3 transcripts. Moreover, using the method presented here, we have also observed alterations in the relative levels of m6A in specific motifs of SOCS genes in celiac disease patients and in pancreatic ß-cells exposed to inflammatory stimuli.


Assuntos
Regiões 3' não Traduzidas , Adenosina/análogos & derivados , Desoxirribonuclease BamHI/química , Motivos de Nucleotídeos , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Adenosina/genética , Adenosina/metabolismo , Células CACO-2 , Humanos , Metilação , Proteína 1 Supressora da Sinalização de Citocina/genética , Proteína 1 Supressora da Sinalização de Citocina/metabolismo , Proteína 3 Supressora da Sinalização de Citocinas/genética , Proteína 3 Supressora da Sinalização de Citocinas/metabolismo
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.
Front Nutr ; 6: 187, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31921880

RESUMO

Celiac disease (CD) patients present a loss of intestinal barrier function due to structural alterations in the tight junction (TJ) network, the most apical unions between epithelial cells. The association of TJ-related gene variants points to an implication of this network in disease susceptibility. This work aims to characterize the functional implication of TJ-related, disease-associated loci in CD pathogenesis. We performed an association study of 8 TJ-related gene variants in a cohort of 270 CD and 91 non-CD controls. The expression level of transcripts located in the associated SNP region was analyzed by RT-PCR in several human tissues and in duodenal biopsies of celiac patients and non-CD controls. (si)RNA-driven silencing combined with gliadin in the Caco2 intestinal cell line was used to analyze the implication of transcripts from the associated region in the regulation of TJ genes. We replicated the association of rs6962966*A variant [p = 0.0029; OR = 1.88 (95%1.24-2.87)], located in an intron of TJ-related MAGI2 coding gene and upstream of RP4-587D13.2 transcript, bioinformatically classified as a long non-coding RNA (lncRNA). The expression of both genes is correlated and constitutively downregulated in CD intestine. Silencing of lncRNA decreases the levels of MAGI2 protein. At the same time, silencing of MAGI2 affects the expression of several TJ-related genes. The associated region is functionally altered in disease, probably affecting CD-related TJ genes.

17.
J Vis Exp ; (137)2018 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-30059028

RESUMO

The purpose of this protocol is to fractionate human intestinal tissue obtained by endoscopy into nuclear and cytoplasmic compartments for the localization analysis of specific proteins or protein complexes in different tissue states (i.e., healthy vs. disease). This method is useful for the fractionation of both fresh and frozen intestinal tissue samples; it is easily accessible for all laboratories and not time consuming.


Assuntos
Conteúdo Gastrointestinal/química , Congelamento , Humanos
18.
Rev Esp Enferm Dig ; 110(7): 458-461, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-29722267

RESUMO

Coeliac disease (CD) is a chronic autoimmune enteropathy triggered by gluten and related prolamines in genetically predisposed individuals. Although CD is a polygenic disease, there is a strong association with genes of the human leukocyte antigen (HLA) region. Most patients present the HLA-DQ2 heterodimer, specifically the DQ2.5 isoform, which is present in around 90-96% of patients of European ancestry.


Assuntos
Doença Celíaca/diagnóstico , Doença Celíaca/genética , Antígenos HLA/genética , Predisposição Genética para Doença , Humanos
19.
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.

20.
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
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