A Strategy for Studying Epigenetic Diversity in Natural Populations: Proof of Concept in Poplar and Oak.

These last 20 years, several techniques have been developed for quantifying DNA methylation, the most studied epigenetic marks in eukaryotes, including the gold standard method, whole-genome bisulphite sequencing (WGBS). WGBS quantifies genome-wide DNA methylation but has several inconveniences rendering it less suitable for population-scale epigenetic studies. The high cost of deep sequencing and the large amounts of data generated prompted us to seek an alternative approach. Restricting studies to parts of the genome would be a satisfactory alternative had there not been a major limitation: the need to select upstream targets corresponding to differentially methylated regions (DMRs) as targets. Given the need to study large numbers of samples, we propose a strategy for investigating DNA methylation variation in natural populations, considering the structural complexity of the genomes with their size and their content in unique as coding regions versus repeated regions as transposable elements. We first identified regions of highly variable DNA methylation in a representative subset of genotypes representative of the biological diversity in the population by WGBS. We then analysed the variations of DNA methylation in these targeted regions at the population level by Sequencing Capture Bisulphite (SeqCapBis). The entire strategy was then validated by applying it to another species. Our strategy was developed as a proof of concept on natural populations of two forest species: Populus nigra and Quercus petraea.

Gata3 hypermethylation and Foxp3 hypomethylation are associated with sustained protection and bystander effect following epicutaneous immunotherapy in peanut-sensitized mice.

BACKGROUND: Epicutaneous immunotherapy (EPIT) is a promising method for treating food allergies. In animal models, EPIT induces sustained unresponsiveness and prevents further sensitization mediated by Tregs. Here, we elucidate the mechanisms underlying the therapeutic effect of EPIT, by characterizing the kinetics of DNA methylation changes in sorted cells from spleen and blood and by evaluating its persistence and bystander effect compared to oral immunotherapy (OIT). METHODS: BALB/c mice orally sensitized to peanut proteins (PPE) were treated by EPIT using a PPE-patch or by PPE-OIT. Another set of peanut-sensitized mice treated by EPIT or OIT were sacrificed following a protocol of sensitization to OVA. DNA methylation was analyzed during immunotherapy and 8 weeks after the end of treatment in sorted cells from spleen and blood by pyrosequencing. Humoral and cellular responses were measured during and after immunotherapy. RESULTS: Analyses showed a significant hypermethylation of the Gata3 promoter detectable only in Th2 cells for EPIT from the 4th week and a significant hypomethylation of the Foxp3 promoter in CD62L+ Tregs, which was sustained only for EPIT. In addition, mice treated with EPIT were protected from subsequent sensitization and maintained the epigenetic signature characteristic for EPIT. CONCLUSIONS: Our study demonstrates that EPIT leads to a unique and stable epigenetic signature in specific T-cell compartments with downregulation of Th2 key regulators and upregulation of Treg transcription factors, likely explaining the sustainability of protection and the observed bystander effect.

Predicting short-term mortality in patients with acute exacerbation of chronic heart failure: The EAHFE-3D scale. / Predicción de la mortalidad a muy corto plazo de los pacientes con insuficiencia cardiaca crónica agudizada: escala EAHFE-3D.

INTRODUCTION AND OBJECTIVES: Prognostic scales are needed in acute exacerbation of chronic heart failure to detect early mortality. The objective of this study is to create a prognostic scale (scale EAHFE-3D) to stratify the risk of death the very short term. PATIENTS AND METHOD: We used the EAHFE database, a multipurpose, multicenter registry with prospective follow-up currently including 6,597 patients with acute heart failure attended at 34 Spanish Emergency Departments from 2007 to 2014. The following variables were collected: demographic, personal history, data of acute episode and 3-day mortality. The derivation cohort included patients recruited during 2009 and 2011 EAHFE registry spots (n=3,640). The classifying variable was all-cause 3-day mortality. A prognostic scale (3D-EAHFE scale) with the results of the multivariate analysis based on the weight of the OR was created. The 3D-EAHFE scale was validated using the cohort of patients included in 2014 spot (n=2,957). RESULTS: A total of 3,640 patients were used in the derivation cohort and 102 (2.8%) died at 3 days. The final scale contained the following variables (maximum 165 points): age≥75 years (30 points), baseline NYHA III-IV (15 points), systolic blood pressure<110mmHg (20 points), room-air oxygen saturation<90% (30 points), hyponatremia (20 points), inotropic or vasopressor treatment (30 points) and need for noninvasive mechanical ventilation (20 points); with a ROC curve of 0.80 (95% CI 0.76-0.84; P<.001). The validation cohort included 2,957 patients (66 died at 3 days, 2.2%), and the scale obtained a ROC curve of 0.76 (95% CI 0.70-0.82; P<.001). The risk groups consisted of very low risk (0-20 points), low risk (21-40 points), intermediate risk (41-60 points), high risk (61-80 points) and very high risk (>80 points), with a mortality (derivation/validation cohorts) of 0/0.5, 0.8/1.0, 2.9/2.8, 5.5/5.8 and 12.7/22.4%, respectively. CONCLUSIONS: EAHFE-3D scale may help to predict the very short term prognosis of patients with acute heart failure in 5 risk groups.

Upregulation of membrane-bound CD40L on CD4+ T cells in women with primary Sjögren's syndrome.

Epigenetic deregulation of genes encoded on the X chromosome as reported for CD40L in lupus could explain the female predominance of autoimmune diseases. We compared CD40L expression on CD4(+) T cells from primary Sjögren's syndrome (pSS) women and healthy controls and investigated DNA methylation patterns of the promoter and enhancer regions of CD40L. The expression of CD40L on activated CD4(+) T cells was higher in patients with pSS than controls after phorbolmyristate acetate and ionomycin activation (P = 0.02). CD40L mRNA level in CD4(+) T cells did not differ between patients with pSS and controls and was similar in both groups in cultures treated with the demethylating agent 5-azacytidine C. Pyrosequencing analysis revealed no significant differences in methylation profiles between patients and controls. Inducible membrane-bound CD40L on CD4(+) T cells is increased in patients with pSS but was not related to epigenetic deregulation by demethylation patterns of the regulatory regions of CD40L.

A polymorphism in the TH 2 locus control region is associated with changes in DNA methylation and gene expression.

BACKGROUND: Genomewide association and epigenetic studies found a region within the RAD50 gene on chromosome 5q31 to be associated with total serum IgE levels and asthma. In mice, this region harbors a locus control region for nearby TH 2 cytokines, which is characterized by four Rad50 DNase I hypersensitive sites (RHS4-7). Among these, RHS7 seems to have the strongest impact on TH 2 differentiation. We investigated whether within the human homolog of RHS7, functional polymorphisms exist, which could affect DNA methylation or gene expression in the 5q31 locus and might have an influence on asthma status or IgE regulation. METHODS: The human RHS7 region was fine mapped using 1000 genomes database information. In silico analysis and electrophoretic mobility shift assays were used to assess SNP function. Allele-specific effects on DNA methylation were evaluated in cord blood (n = 73) and at age of 4.5 years (n = 61) by pyrosequencing. Allele-specific effects on RAD50, IL4, and IL13 expression were analyzed in 100 subjects. Associations with asthma and IgE levels were investigated in the MAGICS/ISAAC II population (n = 1145). RESULTS: Polymorphism rs2240032 in the RHS7 region is suggestive of allele-specific transcription factor binding, affects methylation of the IL13 promoter region and influences RAD50 and IL4 expression (lowest P = 0.0027). It is also associated with total serum IgE levels (P = 0.0227). CONCLUSION: A functional relevant polymorphism in the TH 2 locus control region, equivalent to RHS7 in mice, affects DNA methylation and gene expression within 5q31 and influences total serum IgE on the population level.

MeQA: a pipeline for MeDIP-seq data quality assessment and analysis.

MOTIVATION: We present a pipeline for the pre-processing, quality assessment, read distribution and methylation estimation for methylated DNA immunoprecipitation (MeDIP)-sequence datasets. This is the first MeDIP-seq-specific analytic pipeline that starts at the output of the sequencers. This pipeline will reduce the data analysis load on staff and allows the easy and straightforward analysis of sequencing data for DNA methylation. The pipeline integrates customized scripting and several existing tools, which can deal with both paired and single end data. AVAILABILITY: The package and extensive documentation, and comparison to public data is available at http://life.tongji.edu.cn/meqa/.

Farm exposure and time trends in early childhood may influence DNA methylation in genes related to asthma and allergy.

BACKGROUND: Genetic susceptibility and environmental influences are important contributors to the development of asthma and atopic diseases. Epigenetic mechanisms may facilitate gene by environment interactions in these diseases. METHODS: We studied the rural birth cohort PASTURE (Protection against allergy: study in rural environments) to investigate (a) whether epigenetic patterns in asthma candidate genes are influenced by farm exposure in general, (b) change over the first years of life, and (c) whether these changes may contribute to the development of asthma. DNA was extracted from cord blood and whole blood collected at the age of 4.5 years in 46 samples per time point. DNA methylation in 23 regions in ten candidate genes (ORMDL1, ORMDL2, ORMDL3, CHI3L1, RAD50, IL13, IL4, STAT6, FOXP3, and RUNX3) was assessed by pyrosequencing, and differences between strata were analyzed by nonparametric Wilcoxon-Mann-Whitney tests. RESULTS: In cord blood, regions in ORMDL1 and STAT6 were hypomethylated in DNA from farmers' as compared to nonfarmers' children, while regions in RAD50 and IL13 were hypermethylated (lowest P-value (STAT6) = 0.001). Changes in methylation over time occurred in 15 gene regions (lowest P-value (IL13) = 1.57*10(-8)). Interestingly, these differences clustered in the genes highly associated with asthma (ORMDL family) and IgE regulation (RAD50, IL13, and IL4), but not in the T-regulatory genes (FOXP3, RUNX3). CONCLUSIONS: In this first pilot study, DNA methylation patterns change significantly in early childhood in specific asthma- and allergy-related genes in peripheral blood cells, and early exposure to farm environment seems to influence methylation patterns in distinct genes.

Epigenetic mechanisms and the relationship to childhood asthma.

Epigenetic mechanisms mediate genomic adaption to the environment and epigenetic alterations can contribute to the development of disease phenotypes, as can genetic variants. A variety of molecular mechanisms are involved in epigenetic regulation, including post-transcriptional histone modifications, histone variants, ATP-dependent chromatin remodelling complexes, polycomb/trithorax protein complexes, small and other noncoding RNAs (small interfering RNA and micro RNAs) and DNA methylation. Epigenetic mechanisms have been identified in cancer but very little is known about these effects in complex diseases such as asthma. Epigenetic mechanisms have been found to play a primordial role in T-cell differentiation and novel aspects of asthma and allergy development are now investigated by systematic epigenetic studies. Here we give an introduction to epigenetics, review the existing literature in relation to asthma and asthma-related mechanisms and hypothesise on feasible approaches for the study of epigenetics in asthma in the future.

Epigenetic Changes in Chronic Inflammatory Diseases.

The number of people diagnosed with chronic inflammatory diseases has increased noteworthy in the last 40 years. Spondyloarthritis (SpA), inflammatory bowel diseases (IBD), and psoriasis are the most frequent chronic inflammatory diseases, resulting from a combination of genetic predisposition and environmental factors. Epigenetic modifications include DNA methylation, histone modifications, and small and long noncoding RNAs. They are influenced by environmental exposure, life-style, and aging and have recently been shown to be altered in many complex diseases including inflammatory diseases. While epigenetic modifications have been well characterized in other diseases such as cancer and autoimmune diseases, knowledge on changes in inflammatory diseases is lagging behind with some disease-specific differences. While the DNA methylation profile of different cell types in patients with IBD has been relatively well described, less is known on changes implicated in psoriasis, and no systematic genome-wide studies have so far been performed in SpA. In this chapter, we review in detail the reported changes in patterns of DNA methylation and posttranslational histone modifications in chronic inflammatory diseases highlighting potential connections between disease-associated pathophysiological changes such as the dysbiosis of the microbiome or genetic variations associated with disease susceptibility and the epigenome. We also discuss important parameters of meaningful epigenetic studies such as the use of well defined, disease-relevant cell populations, and elude on the potential future of engineering of the epigenome in inflammatory diseases.

Gene expression profiling in insulinomas of Men1 beta-cell mutant mice reveals early genetic and epigenetic events involved in pancreatic beta-cell tumorigenesis.

Mutations of the MEN1 gene lead to the occurrence of multiple endocrine neoplasia type 1 (MEN1). To gain insights into the mechanisms of the tumorigenesis related to MEN1 inactivation, we have used mice in which the Men1 gene was specifically disrupted in pancreatic beta-cells. In these mice, we observed full penetrance of insulinoma with defined histological characteristics of tumorigenesis. To identify the genetic factors taking part in the tumour development, we performed gene expression profiling analysis of these insulinomas at different stages. Here, we show that in late stage insulinomas, 56 genes are up-regulated and 194 are down-regulated more than fourfold compared with normal pancreatic islets. Clustering analysis reveals the deregulation of Hox gene family and the genes involved in cell proliferation and cell cycle control. The altered expression of Igf2, Igfbp3 and Igfbp6 as well as cyclin A2, B2 and D2 are confirmed by quantitative RT-PCR, with the overexpression of all the three cyclins found in early stage insulinomas. Moreover, an increased proportion of cyclin A2- and D2-expressing cells and the overexpression of insulin-like growth factor 2 (IGF2) protein are detected in mouse Men1 insulinomas by immunostaining. Interestingly, the analysis of DNA methylation patterns by quantitative serial pyrosequencing reveals that four specific CpGs in the intragenic differentially methylated region 2 (DMR2) region of the Igf2 gene known to augment transcription through methylation are significantly hypermethylated in insulinomas of Men1 beta-cell mutant mice at 6 and 10 months of age, even before IGF2 overexpression can be detected. Thus, our data indicate the involvement of both genetic and epigenetic mechanisms in early tumorigenesis of beta-cells related to MEN1 inactivation.

Severe hepatotoxicity due to anti-tuberculosis drugs in Spain.

OBJECTIVES: To determine the frequency of severe hepatotoxicity due to anti-tuberculosis (TB) drugs, and predictors of development of acute liver failure or of death. METHODS: A retrospective study conducted by members of the Spanish Society of Pneumology from 18 hospitals during 1997-2001. A case of severe hepatotoxicity was defined as any asymptomatic patient with a ten-fold increase in transaminases or three-fold increase in colostasis parameters, or, among patients with hepatitis symptoms, any raised hepatic parameters or development of hepatic failure. Predictive factors were studied using logistic regression, calculating odds ratios (OR) and their 95% confidence intervals (CI). RESULTS: One hundred and six patients developed severe hepatotoxicity. Of a total of 3510 patients, 90 were treated for active TB (2.56%). Eleven cases (10.3%) presented with acute liver failure, three of whom underwent liver transplant. The global case fatality rate was 4.7% (five cases, three associated with alcohol use or hepatotoxic drugs). The predictors of poor prognosis were total bilirubin > 2 mg/dl (OR 9.4, 95% CI 1.0-85.5) and serum creatinine > 1.5 mg/dl (OR 32.1, 95% CI 2.4-424.6). CONCLUSIONS: Severe hepatotoxicity due to anti-tuberculosis drugs is associated with a high fatality rate. Prevention should be based on informing patients and frequent clinical and laboratory controls.

Predicción de la mortalidad a muy corto plazo de los pacientes con insuficiencia cardiaca crónica agudizada: escala EAHFE-3D / Predicting short-term mortality in patients with acute exacerbation of chronic heart failure: The EAHFE-3D scale

INTRODUCCIÓN Y OBJETIVOS: Disponer de escalas pronósticas en la insuficiencia cardiaca crónica agudizada para detectar la mortalidad precoz es fundamental. El objetivo de este estudio es crear una escala pronóstica (escala EAHFE-3D) que estratifique el riesgo de muerte a muy corto plazo. PACIENTES Y MÉTODO: Se utilizó el registro EAHFE, multipropósito y multicéntrico, con seguimiento prospectivo que incluye 6.597 pacientes con insuficiencia cardiaca crónica agudizada atendidos en 34 servicios de urgencias españoles entre 2007 y 2014. Se recogieron variables demográficas, antecedentes personales, datos del episodio agudo, destino final y mortalidad a los 3 días. La cohorte de derivación incluye pacientes seleccionados entre 2009 y 2011 en el registro EAHFE (n = 3.640). La variable a estudio fue la mortalidad a los 3 días. Se creó una escala pronóstica (escala EAHFE-3D) con los resultados del estudio multivariante en función del peso de la OR. La escala fue validada utilizando una cohorte de pacientes incluidos en 2014 (n = 2.957). RESULTADOS: Se analizaron 3.640 pacientes (102 muertos a los 3 días, 2,8%) en la cohorte de derivación. La escala final contiene las siguientes variables (máximo 165 puntos): edad≥ 75 años (30 puntos), NYHA basal III-IV (15 puntos), presión arterial sistólica < 110 mmHg (20 puntos), saturación de O2 < 90% (30 puntos), hiponatremia (20 puntos), tratamiento inotropo o vasopresor (30 puntos) y necesidad de ventilación mecánica no invasiva (20 puntos), con un área bajo la curva ROC de 0,80 (IC 95% 0,76-0,84; p < 0,001). La cohorte de validación incluye 2.957 pacientes (66 muertos a los 3 días, 2,2%) y la escala obtiene un área bajo la curva ROC de 0,76 (IC 95% 0,70-0,82; p < 0,001). Los grupos fueron: muy bajo riesgo (0-20 puntos), bajo riesgo (21-40 puntos), riesgo intermedio (41-60 puntos), alto riesgo (61-80 puntos) y muy alto riesgo (> 80 puntos), con una mortalidad (cohorte de derivación/validación) de 0/0,5, 0,8/1,0%, 2,9/2,8, 5,5/5,8 y 12,7/22,4%, respectivamente. CONCLUSIONES: La escala EAHFE-3D puede ser de ayuda para estratificar el pronóstico a muy corto plazo de los pacientes con insuficiencia cardiaca crónica agudizada en 5 grupos de riesgo

INTRODUCTION AND OBJECTIVES: Prognostic scales are needed in acute exacerbation of chronic heart failure to detect early mortality. The objective of this study is to create a prognostic scale (scale EAHFE-3D) to stratify the risk of death the very short term. PATIENTES AND METHOD: We used the EAHFE database, a multipurpose, multicenter registry with prospective follow-up currently including 6,597 patients with acute heart failure attended at 34 Spanish Emergency Departments from 2007 to 2014. The following variables were collected: demographic, personal history, data of acute episode and 3-day mortality. The derivation cohort included patients recruited during 2009 and 2011 EAHFE registry spots (n=3,640). The classifying variable was all-cause 3-day mortality. A prognostic scale (3D-EAHFE scale) with the results of the multivariate analysis based on the weight of the OR was created. The 3D-EAHFE scale was validated using the cohort of patients included in 2014 spot (n=2,957). RESULTS: A total of 3,640 patients were used in the derivation cohort and 102 (2.8%) died at 3 days. The final scale contained the following variables (maximum 165 points): age≥75 years (30 points), baseline NYHA III-IV (15 points), systolic blood pressure < 110mmHg (20 points), room-air oxygen saturation<90% (30 points), hyponatremia (20 points), inotropic or vasopressor treatment (30 points) and need for noninvasive mechanical ventilation (20 points); with a ROC curve of 0.80 (95% CI 0.76-0.84; P < .001). The validation cohort included 2,957 patients (66 died at 3 days, 2.2%), and the scale obtained a ROC curve of 0.76 (95% CI 0.70-0.82; P < .001). The risk groups consisted of very low risk (0-20 points), low risk (21-40 points), intermediate risk (41-60 points), high risk (61-80 points) and very high risk (>80 points), with a mortality (derivation/validation cohorts) of 0/0.5, 0.8/1.0, 2.9/2.8, 5.5/5.8 and 12.7/22.4%, respectively. CONCLUSIONS: EAHFE-3D scale may help to predict the very short term prognosis of patients with acute heart failure in 5 risk groups