Epigenetic regulation of cyclooxygenase-2 by methylation of c8orf4 in pulmonary fibrosis

Fibroblasts derived from the lungs of patients with idiopathic pulmonary fibrosis (IPF) and systemic sclerosis (SSc) produce low levels of prostaglandin (PG) E(2), due to a limited capacity to up-regulate cyclooxygenase-2 (COX-2). This deficiency contributes functionally to the fibroproliferative state, however the mechanisms responsible are incompletely understood. In the present study, we examined whether the reduced level of COX-2 mRNA expression observed in fibrotic lung fibroblasts is regulated epigenetically. The DNA methylation inhibitor, 5-aza-2′-deoxycytidine (5AZA) restored COX-2 mRNA expression by fibrotic lung fibroblasts dose dependently. Functionally, this resulted in normalization of fibroblast phenotype in terms of PGE(2) production, collagen mRNA expression and sensitivity to apoptosis. COX-2 methylation assessed by bisulfite sequencing and methylation microarrays was not different in fibrotic fibroblasts compared with controls. However, further analysis of the methylation array data identified a transcriptional regulator, chromosome 8 open reading frame 4 (thyroid cancer protein 1, TC-1) (c8orf4), which is hypermethylated and down-regulated in fibrotic fibroblasts compared with controls. siRNA knockdown of c8orf4 in control fibroblasts down-regulated COX-2 and PGE(2) production generating a phenotype similar to that observed in fibrotic lung fibroblasts. Chromatin immunoprecipitation demonstrated that c8orf4 regulates COX-2 expression in lung fibroblasts through binding of the proximal promoter. We conclude that the decreased capacity of fibrotic lung fibroblasts to up-regulate COX-2 expression and COX-2-derived PGE(2) synthesis is due to an indirect epigenetic mechanism involving hypermethylation of the transcriptional regulator, c8orf4

DNA Methylome Alterations are Associated with Airway Macrophage Differentiation and Phenotype During Lung Fibrosis

RATIONALE: Airway macrophages (AMs) are key regulators of the lung environment and are implicated in the pathogenesis of idiopathic pulmonary fibrosis (IPF), a fatal respiratory disease with no cure. However, knowledge of epigenetics of AMs in IPF are limited. METHODS: We undertook DNA methylation profiling using Illumina EPIC (850k) arrays in sorted AMs from Healthy (n=14) and IPF (n=30) donors. Cell-type deconvolution was performed using reference myeloid-cell DNA methylomes. MEASUREMENTS AND MAIN RESULTS: Our analysis revealed epigenetic heterogeneity was a key characteristic of IPF-AMs. DNAm 'clock' analysis indicated epigenetic alterations in IPF-AMs was not associated with accelerated ageing. In differential DNAm analysis, we identified numerous differentially methylated positions (DMPs, n=11) and regions (DMRs, n=49) between healthy and IPF AMs respectively. DMPs and DMRs encompassed genes involved in lipid (LPCAT1) and glucose (PFKFB3) metabolism and importantly, DNAm status was associated with disease severity in IPF. CONCLUSIONS: Collectively, our data identify that changes in the epigenome are associated with development and function of AMs in the IPF lung

Assessment of funnel plot asymmetry and publication bias in reproductive health meta-analyses: an analytic survey

BACKGROUND: Despite efforts to assure high methodological standards, systematic reviews may be affected by publication bias. The objective of this study was to evaluate the occurrence of publication bias in a collection of high quality systematic reviews on reproductive health. METHODS: Systematic reviews included in the Reproductive Health Library (RHL), issue No 9, were assessed. Funnel plot was used to assess meta-analyses containing 10 or more trials reporting a binary outcome. A funnel plot, the estimated number of missing studies and the adjusted combined effect size were obtained using the "trim and fill method". Meta-analyses results that were not considered to be robust due to a possible publication bias were submitted to a more detailed assessment. RESULTS: A total of 21 systematic reviews were assessed. The number of trials comprising each one ranged from 10 to 83 (median = 13), totaling 379 trials, whose results have been summarized. None of the reviews had reported any evaluation of publication bias or funnel plot asymmetry. Some degree of asymmetry in funnel plots was observed in 18 of the 21 meta-analyses evaluated (85.7%), with the estimated number of missing studies ranging from 1 to 18 (median = 3). Only for three meta-analyses, the conclusion could not be considered robust due to a possible publication bias. CONCLUSION: Asymmetry is a frequent finding in funnel plots of meta-analyses in reproductive health, but according to the present evaluation, less than 15% of meta-analyses report conclusions that would not be considered robust. Publication bias and other sources of asymmetry in funnel plots should be systematically addressed by reproductive health meta-analysts. Next amendments in Cochrane systematic reviews should include this type of evaluation. Further studies regarding the evolution of effect size and publication bias over time in systematic reviews in reproductive health are needed

Selfish mutations dysregulating RAS-MAPK signaling are pervasive in aged human testes

Mosaic mutations present in the germline have important implications for reproductive risk and disease transmission. We previously demonstrated a phenomenon occurring in the male germline, whereby specific mutations arising spontaneously in stem cells (spermatogonia) lead to clonal expansion, resulting in elevated mutation levels in sperm over time. This process, termed selfish spermatogonial selection, explains the high spontaneous birth prevalence and strong paternal age-effect of disorders such as achondroplasia, Apert, Noonan and Costello syndromes, with direct experimental evidence currently available for specific positions of six genes (FGFR2, FGFR3, RET, PTPN11, HRAS and KRAS). We present a discovery screen to identify novel mutations and genes showing evidence of positive selection in the male germline, by performing massively parallel simplex PCR using RainDance technology to interrogate mutational hotspots in 67 genes (51.5 kb in total) in 276 biopsies of testes from 5 men (median age: 83 years). Following ultra-deep sequencing (~16,000x), development of a low-frequency variant prioritization strategy and targeted validation, we identified 61 distinct variants present at frequencies as low as 0.06%, including 54 variants not previously directly associated with selfish selection. The majority (80%) of variants identified have previously been implicated in developmental disorders and/or oncogenesis and include mutations in six newly associated genes (BRAF, CBL, MAP2K1, MAP2K2, RAF1 and SOS1), all of which encode components of RAS-MAPK pathway and activate signaling. Our findings extend the link between mutations dysregulating the RAS-MAPK pathway and selfish selection, and show that the ageing male germline is a repository for such deleterious mutations

Pulmonary hypertension in interstitial lung disease: Limitations of echocardiography compared to cardiac catheterization

BACKGROUND AND OBJECTIVE: In interstitial lung disease (ILD), pulmonary hypertension (PH) is a major adverse prognostic determinant. Transthoracic echocardiography (TTE) is the most widely used tool when screening for PH, although discordance between TTE and right heart catheter (RHC) measured pulmonary haemodynamics is increasingly recognized. We evaluated the predictive utility of the updated European Society of Cardiology/European Respiratory Society (ESC/ERS) TTE screening recommendations against RHC testing in a large, well-characterized ILD cohort. METHODS: Two hundred and sixty-five consecutive patients with ILD and suspected PH underwent comprehensive assessment, including RHC, between 2006 and 2012. ESC/ERS recommended tricuspid regurgitation (TR) velocity thresholds for assigning high (>3.4 m/s), intermediate (2.9-3.4 m/s) and low (3.4 m/s, and excluded PH in 60% of ILD subjects with a TR velocity <2.8 m/s. Thus, the ESC/ERS guidelines misclassified 40% of subjects as 'low probability' of PH, when PH was confirmed on subsequent RHC. Evaluating alternative TR velocity thresholds for assigning a low probability of PH did not significantly improve the ability of TR velocity to exclude a diagnosis of PH. CONCLUSION: In patients with ILD and suspected PH, currently recommended ESC/ERS TR velocity screening thresholds were associated with a high positive predictive value (86%) for confirming PH, but were of limited value in excluding PH, with 40% of patients misclassified as low probability when PH was confirmed at subsequent RHC

Sitting time and obesity in a sample of adults from Europe and the USA

This is an Accepted Manuscript of an article published by Taylor & Francis in Annals of Human Biology on 25 Sep 2016, available online: http://www.tandfonline.com/10.1080/03014460.2016.1232749.Obesity is a risk factor for many chronic diseases and the prevalence is increasing worldwide. Research suggests that sedentary behaviour (sitting) may be related to obesity.To examine the association between sitting time and obesity, while controlling for physical activity, in a large international sample.5338 adults from the UK, USA, Germany, Spain, Italy, France, Portugal, Austria and Switzerland self-reported their total daily sitting time, physical activity, age, height and weight. BMI (kg/m(2)), total physical activity (MET-minutes/week) and sitting time (hours/day) were derived. Participants were grouped into quartiles based on their daily sitting time (<4, 4 - ≤6, 6 - ≤8, and >8 hours/day) and logistic regression models explored the odds of being obese versus normal weight for each sitting time quartile.Participants in the highest sitting time quartile (≥8 hours/day) had 62% higher odds of obesity compared to participants in the lowest quartile (<4 hours/day) after adjustment for physical activity and other confounding variables (OR = 1.62, 95% CI = 1.24-2.12, p<0.01).Sitting time is associated with obesity in adults, independent of physical activity. Future research should clarify this association using objective measures of sitting time and physical activity to further inform health guidelines

Sources of variability in childhood obesity indicators and related behaviors

The purpose of this study was to describe sources of variability in obesity-related variables in 6022 children aged 9-11 years from 12 countries. The study design involved recruitment of students, nested within schools, which were nested within study sites. Height, weight and waist circumference (WC) were measured and body mass index (BMI) was calculated; sleep duration and total and in-school moderate-to-vigorous physical activity (MVPA) and sedentary time were measured by accelerometry; and diet scores were obtained by questionnaire. Variance in most variables was largely explained at the student level: BMI (91.9%), WC (93.5%), sleep (75.3%), MVPA (72.5%), sedentary time (76.9%), healthy diet score (88.3%), unhealthy diet score (66.2%), with the exception of in-school MVPA (53.8%) and in-school sedentary time (25.1%). Variance explained at the school level ranged from 3.3% for BMI to 29.8% for in-school MVPA, and variance explained at the site level ranged from 3.2% for WC to 54.2% for in-school sedentary time. In general, more variance was explained at the school and site levels for behaviors than for anthropometric traits. Given the variance in obesity-related behaviors in primary school children explained at school and site levels, interventions that target policy and environmental changes may enhance obesity intervention efforts.Peer reviewe