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List of genes exhibiting loss of promoter core 5hmC and gain of 5mC in PB driven liver tumours

Describes more detailed methods employed in main manuscript and supplementary data.

17 Supplementary Figures supporting the data and interpretation presented in the main manuscript.

Lists of genes transcritpionally altered following chronic PB exposure and in the resulting liver tumours (see figure 4a)

Objective To examine the association between gestational age, telomere length (TL) and rate of shortening in newborns. Study design Genomic DNA was isolated from buccal samples of 39 term infants at birth and one year and 32 preterm infants at birth, term-adjusted age (40 weeks post-conception) and age one-year corrected for gestational duration. Telomere length was measured by quantitative real-time PCR. Demographic and clinical data were collected during clinic or research visits and from hospital records. Socioeconomic status was estimated using the deprivation category (DEPCAT) scores derived from the Carstairs score of the subject’s postal code. Results At birth, preterm infants had longer telomeres than infants born at term. However, there was no difference in telomere length between preterm infants and term infants at one year of age, implying that the rate of telomere shortening was greater in pre-term than term infants. Interestingly, TL at age 40 weeks post-conception in preterm infants was significantly longer than term infant TL at birth, suggesting that time since conception is not the only factor that affects rate of shortening. Several factors, including sex, fetal growth restriction, maternal age, maternal booking body mass index (BMI), mother education level and DEPCAT score, also differed between the preterm and term groups. Conclusions Preterm infants have longer telomeres than term infants at birth. In the studied cohort, the rate of telomere shortening was greater in the premature group compared with the term infants. This finding agrees with previous studies using cord blood, suggesting that the longer TL in premature infants detected at birth do not persist and demonstrating that use of saliva DNA is acceptable for studies of telomere dynamics in infants. However, that the TL at age 40 weeks post-conception in preterm is longer than term infants at birth suggests that biological factors other than time since conception also affect rate of shortening.

Highlights • 60 studies of association between DNAm and human brain MRI were identified. • Differential DNAm is associated with brain structure and function throughout life. • There is modest consistency between DNAm and image endophenotypes. • Approaches for reducing heterogeneity in DNAm-MRI analyses are proposed., MRI has enhanced our capacity to understand variations in brain structure and function conferred by the genome. We identified 60 studies that report associations between DNA methylation (DNAm) and human brain structure/function. Forty-three studies measured candidate loci DNAm; seventeen measured epigenome-wide DNAm. MRI features included region-of-interest and whole-brain structural, diffusion and functional imaging features. The studies report DNAm-MRI associations for: neurodevelopment and neurodevelopmental disorders; major depression and suicidality; alcohol use disorder; schizophrenia and psychosis; ageing, stroke, ataxia and neurodegeneration; post-traumatic stress disorder; and socio-emotional processing. Consistency between MRI features and differential DNAm is modest. Sources of bias: variable inclusion of comparator groups; different surrogate tissues used; variation in DNAm measurement methods; lack of control for genotype and cell-type composition; and variations in image processing. Knowledge of MRI features associated with differential DNAm may improve understanding of the role of DNAm in brain health and disease, but caution is required because conventions for linking DNAm and MRI data are not established, and clinical and methodological heterogeneity in existing literature is substantial.

Impaired social function is a core feature of many psychiatric illnesses. Adverse experiences during childhood increase risk for mental illness, however it is currently unclear whether stress early in life plays a direct role in the development of social difficulties. Using a rat model of pre-pubertal stress (PPS), we investigated effects on social behaviour, oxytocin and arginine vasopressin (AVP) in the periphery (plasma) and centrally in the paraventricular and supraoptic hypothalamic nuclei. We also explored social performance and AVP expression (plasma) in participants with borderline personality disorder (BPD) who experienced a high incidence of childhood stress. Social behaviour was impaired and AVP expression increased in animals experiencing PPS and participants with BPD. Behavioural deficits in animals were rescued through administration of the AVPR1a antagonist Relcovaptan (SR49059). AVP levels and recognition of negative emotions were significantly correlated in BPD participants only. In conclusion, early life stress plays a role in the precipitation of social dysfunction, and AVP mediates at least part of this effect.

Context Remission rates in young people with Graves hyperthyroidism are less than 25% after 2 years of thionamide antithyroid drug (ATD). Objective We explored whether rituximab (RTX), a B-lymphocyte–depleting agent, would increase remission rates when administered with a short course of ATD. Methods This was an open-label, multicenter, single-arm, phase 2 trial in young people (ages, 12-20 years) with Graves hyperthyroidism. An A’Hern design was used to distinguish an encouraging remission rate (40%) from an unacceptable rate (20%). Participants presenting with Graves hyperthyroidism received 500 mg RTX and 12 months of ATD titrated according to thyroid function. ATDs were stopped after 12 months and primary outcome assessed at 24 months. Participants had relapsed at 24 months if thyrotropin was suppressed and free 3,5,3′-triiodothyronine was raised; they had received ATD between months 12 and 24; or they had thyroid surgery/radioiodine. Results A total of 27 participants were recruited and completed the trial with no serious side effects linked to treatment. Daily carbimazole dose at 12 months was less than 5 mg in 21 of 27 participants. Thirteen of 27 participants were in remission at 24 months (48%, 90% one-sided CI, 35%-100%); this exceeded the critical value (9) for the A’Hern design and provided evidence of a promising remission rate. B-lymphocyte count at 28 weeks, expressed as a percentage of baseline, was related to likelihood of remission. Conclusion Adjuvant RTX, administered with a 12-month course of ATD, may increase the likelihood of remission in young people with Graves hyperthyroidism. A randomized trial of adjuvant RTX in young people with Graves hyperthyroidism is warranted.

Exposure to early life stress (ELS) during childhood or prenatally increases the risk of future psychiatric disorders. The effect of stress exposure during the neonatal period is less well understood. In preterm infants, exposure to invasive procedures is associated with altered brain development and future stress responses suggesting that the neonatal period could be a key time for the programming of mental health. Previous studies suggest that ELS affects the hypothalamic epigenome, making it a good candidate to mediate these effects. In this study, we used a mouse model of early life stress (modified maternal separation; MMS). We hypothesised MMS would affect the hypothalamic transcriptome and DNA methylome, and impact on adult behaviour. MMS involved repeated stimulation of pups for 1.5 h/day, whilst separated from their mother, from postnatal day (P) 4–6. 3’mRNA sequencing and DNA methylation immunoprecipitation (meDIP) sequencing were performed on hypothalamic tissue at P6. Behaviour was assessed with the elevated plus, open field mazes and in-cage monitoring at 3–4 months of age. MMS was only associated with subtle changes in gene expression, but there were widespread alterations in DNA methylation. Notably, differentially methylated regions were enriched for synapse-associated loci. MMS resulted in hyperactivity in the elevated plus and open field mazes, but in-cage monitoring revealed that this was not representative of habitual hyperactivity. ELS has marked effects on DNA methylation in the hypothalamus in early life and results in stress-specific hyperactivity in young adulthood. These results have implications for the understanding of ELS-mediated effects on brain development. Supplementary Information The online version contains supplementary material available at 10.1186/s13072-021-00405-8.

Metformin is widely used in pregnancy, despite lack of long-term safety for children. We hypothesised that metformin exposure in utero is associated with increased cardiovascular risk. We tested this hypothesis in a follow-up study of children born to obese mothers who had participated in a randomised controlled trial of metformin versus placebo in pregnancy (EMPOWaR). We measured body composition, peripheral blood pressure (BP), arterial pulse wave velocity and central haemodynamics (central BP and augmentation index) using an oscillometric device in 40 children of mean (SD) age 5.78 (0.93) years, exposed to metformin (n = 19) or placebo (n = 21) in utero. There were no differences in any of the anthropometric or vascular measures between metformin and placebo-exposed groups in univariate analyses, or after adjustment for potential confounders including the child’s behaviour, diet and activity levels. Post-hoc sample size calculation indicated we would have detected large clinically significant differences between the groups but would need an unfeasible large number to detect possible subtle differences in key cardiovascular risk parameters in children at this age of follow-up. Our findings suggest no evidence of increased cardiovascular risk in children born to obese mothers who took metformin in pregnancy and increase available knowledge of the long-term safety of metformin on childhood outcomes.

Epigenetic regulation of gene expression is critical for normal development. Dysregulation of the epigenome can lead to the development and progression of a number of diseases relevant to paediatricians, including disorders of genomic imprinting and malignancies. It has long been recognised that early life events have implications for future disease risk, and epigenetic modifications may play a role in this, although further high-quality research is needed to better understand the underlying mechanisms. Research in the field of epigenetics will contribute to a greater understanding of growth, development and disease; however, paediatricians need to be able to interpret such research critically, in order to use the potential advances brought about through epigenetic studies while appreciating their limitations.

Neonatal encephalopathy due to hypoxia–ischemia is associated with adverse neurodevelopmental effects. The involvement of branched chain amino acids (BCAAs) in this is largely unexplored. Transport of BCAAs at the plasma membrane is facilitated by SLC7A5/SLC3A2, which increase with hypoxia. We hypothesized that hypoxia would alter BCAA transport and metabolism in the neonatal brain. We investigated this using an organotypic forebrain slice culture model with, the SLC7A5/SLC3A2 inhibitor, 2-Amino-2-norbornanecarboxylic acid (BCH) under normoxic or hypoxic conditions. We subsequently analysed the metabolome and candidate gene expression. Hypoxia was associated with increased expression of SLC7A5 and SLC3A2 and an increased tissue abundance of BCAAs. Incubation of slices with 13C-leucine confirmed that this was due to increased cellular uptake. BCH had little effect on metabolite abundance under normoxic or hypoxic conditions. This suggests hypoxia drives increased cellular uptake of BCAAs in the neonatal mouse forebrain, and membrane mediated transport through SLC7A5 and SLC3A2 is not essential for this process. This indicates mechanisms exist to generate the compounds required to maintain essential metabolism in the absence of external nutrient supply. Moreover, excess BCAAs have been associated with developmental delay, providing an unexplored mechanism of hypoxia mediated pathogenesis in the developing forebrain.

Summary This protocol describes the production of hepatocyte-like cells (HLCs) from human pluripotent stem cells and how to induce hepatic steatosis, a condition characterized by intracellular lipid accumulation. Following differentiation to an HLC phenotype, intracellular lipid accumulation is induced with a steatosis induction cocktail, allowing the user to examine the cellular processes that underpin hepatic steatosis. Furthermore, the renewable nature of our system, on a defined genetic background, permits in-depth mechanistic analysis, which may facilitate therapeutic target identification in the future. For complete details on the use and execution of this protocol, please refer to Sinton et al. (2021)., Graphical abstract, Highlights • A detailed protocol to produce stem cell-derived hepatocyte-like cells • Lactate, pyruvate, and octanoate treatment induces steatosis in hepatocyte-like cells • A reliable tool for examining the processes that underpin hepatic steatosis, This protocol describes the production of hepatocyte-like cells (HLCs) from human pluripotent stem cells and how to induce hepatic steatosis, a condition characterized by intracellular lipid accumulation. Following differentiation to an HLC phenotype, intracellular lipid accumulation is induced with a steatosis induction cocktail, allowing the user to examine the cellular processes that underpin hepatic steatosis. Furthermore, the renewable nature of our system, on a defined genetic background, permits in-depth mechanistic analysis, which may facilitate therapeutic target identification in the future.

Lasting effects of adversity, such as exposure to childhood adversity (CA) on disease risk, may be embedded via epigenetic mechanisms but findings from human studies investigating the main effects of such exposure on epigenetic measures, including DNA methylation (DNAm), are inconsistent. Studies in perinatal tissues indicate that variability of DNAm at birth is best explained by the joint effects of genotype and prenatal environment. Here, we extend these analyses to postnatal stressors. We investigated the contribution of CA, cis genotype (G), and their additive (G + CA) and interactive (G × CA) effects to DNAm variability in blood or saliva from five independent cohorts with a total sample size of 1074 ranging in age from childhood to late adulthood. Of these, 541 were exposed to CA, which was assessed retrospectively using self-reports or verified through social services and registries. For the majority of sites (over 50%) in the adult cohorts, variability in DNAm was best explained by G + CA or G × CA but almost never by CA alone. Across ages and tissues, 1672 DNAm sites showed consistency of the best model in all five cohorts, with G × CA interactions explaining most variance. The consistent G × CA sites mapped to genes enriched in brain-specific transcripts and Gene Ontology terms related to development and synaptic function. Interaction of CA with genotypes showed the strongest contribution to DNAm variability, with stable effects across cohorts in functionally relevant genes. This underscores the importance of including genotype in studies investigating the impact of environmental factors on epigenetic marks.

Summary Nonalcoholic fatty liver disease (NAFLD) is currently the most prevalent form of liver disease worldwide. This term encompasses a spectrum of pathologies, from benign hepatic steatosis to non-alcoholic steatohepatitis, which have, to date, been challenging to model in the laboratory setting. Here, we present a human pluripotent stem cell (hPSC)-derived model of hepatic steatosis, which overcomes inherent challenges of current models and provides insights into the metabolic rewiring associated with steatosis. Following induction of macrovesicular steatosis in hepatocyte-like cells using lactate, pyruvate, and octanoate (LPO), respirometry and transcriptomic analyses revealed compromised electron transport chain activity. 13C isotopic tracing studies revealed enhanced TCA cycle anaplerosis, with concomitant development of a compensatory purine nucleotide cycle shunt leading to excess generation of fumarate. This model of hepatic steatosis is reproducible, scalable, and overcomes the challenges of studying mitochondrial metabolism in currently available models., Graphical Abstract, Highlights • LPO-exposed hPSC-derived hepatocyte-like cells resemble human hepatic steatosis • Steatosis is associated with decreased mitochondrial maximal respiration • Purine nucleotide cycle activity increases in steatotic hepatocytes • Excess fumarate production is associated with induction of macrovesicular steatosis, Endocrine System Physiology; Stem Cells Research; Transcriptomics

An optimal early life environment is crucial for ensuring ideal neurodevelopmental outcomes. Brain development consists of a finely tuned series of spatially and temporally constrained events, which may be affected by exposure to a sub-optimal intra-uterine environment. Evidence suggests brain development may be particularly vulnerable to factors such as maternal nutrition, infection and stress during pregnancy. In this review, we discuss how maternal factors such as these can affect brain development and outcome in offspring, and we also identify evidence which suggests that the outcome can, in many cases, be stratified by socio-economic status (SES), with individuals in lower brackets typically having a worse outcome. We consider the relevant epidemiological evidence and draw parallels to mechanisms suggested by preclinical work where appropriate. We also discuss possible transgenerational effects of these maternal factors and the potential mechanisms involved. We conclude that modifiable factors such as maternal nutrition, infection and stress are important contributors to atypical brain development and that SES also likely has a key role.

SummaryNonalcoholic fatty liver disease (NAFLD) affects ~88% of obese individuals and is characterised by hepatic lipid accumulation. Mitochondrial metabolic dysfunction is a feature of NAFLD. We used a human pluripotent stem cell-based system to determine how mitochondrial dysfunction is linked to hepatic lipid accumulation. We induced lipid accumulation in hepatocyte-like cells (HLCs) using lactate, pyruvate and octanoate (LPO). Transcriptomic analysis revealed perturbation of mitochondrial respiratory pathways in LPO exposed cells. Using13C isotopic tracing, we identified truncation of the TCA cycle in steatotic HLCs. We show that increased purine nucleotide cycle (PNC) activity fuels fumarate accumulation and drives lipid accumulation in steatotic cells. These findings provide new insights into the pathogenesis of hepatic steatosis and may lead to an improved understanding of the metabolic and transcriptional rewiring associated with NAFLD.

Early life stress during childhood is associated with a number of psychiatric disorders that manifest across the life course. Preterm birth is a profound stressor, and an important cause of cognitive impairment, as well as neurodevelopmental and psychiatric disorders. However, the mechanisms that link events during the early neonatal period with later functional problems are poorly understood. We developed a novel mouse model of early life stress (modified maternal separation; MMS) with specific relevance to preterm birth (PTB) and hypothesised it would affect the hypothalamic transcriptome and DNA methylome and impact on behaviour in adulthood. MMS consisted of repeatedly stimulating pups for 1.5 hours/day, whilst separated from their mother, from postnatal day (P)4-6. 3’ RNA sequencing and DNA methylation immunoprecipitation (meDIP) sequencing was performed on the hypothalamus at P6. Behaviour was assessed with the elevated plus and open field mazes, and in-cage monitoring at 3-4 months of age. Although MMS was only associated with subtle changes in gene expression there were widespread alterations in DNA methylation. Notably, differentially methylated regions were enriched for synapse-associated loci. MMS also resulted in hyperactivity in the elevated plus and open field mazes, but in-cage monitoring revealed that this was not representative of habitual hyperactivity. In conclusion we describe a novel model of early life stress with relevance to PTB, with marked effects on DNA methylation in the hypothalamus and with stress-specific hyperactivity in young adulthood. We suggest that these results have implications for the understanding of early life stress mediated effects on brain development.

Nonalcoholic fatty liver disease (NAFLD) is currently the most prevalent form of liver disease worldwide. This term covers a spectrum of pathologies, from benign hepatic steatosis to non-alcoholic steatohepatitis (NASH). As the disease progresses, NASH can develop into cirrhosis and hepatocellular carcinoma. However, the underlying mechanisms and the factors which predispose an individual to disease progression remain poorly understood. Whilst NAFLD appears to be associated with mitochondrial dysfunction, it is unclear whether this is due to respiratory impairment, changes in mitochondrial mass, or mitochondrial fragmentation. Using a human pluripotent stem cell-based model of NAFLD we show that exposure to lactate, pyruvate and octanoic acid results in the development of macrovesicular steatosis. We do not observe changes in mitochondrial mass or fragmentation but do find decreases in maximal respiration and reserve capacity, suggesting impairment in the electron transport chain (ETC). Taken together, these findings indicate that the development of macrovesicular steatosis in NAFLD may be linked to the impairment of the ETC in mitochondria.

IntroductionPreterm birth is closely associated with altered brain development and is a leading cause of neurodevelopmental, cognitive and behavioural impairments across the life course. We aimed to investigate neuroanatomic variation and adverse outcomes associated with preterm birth by studying a cohort of preterm infants and controls born at term using brain MRI linked to biosamples and clinical, environmental and neuropsychological data.Methods and analysisTheirworld Edinburgh Birth Cohort is a prospective longitudinal cohort study at the University of Edinburgh. We plan to recruit 300 infants born at Ethics and disseminationEthical approval has been obtained from the National Research Ethics Service (NRES), South East Scotland Research Ethics Committee (NRES numbers 11/55/0061 and 13/SS/0143 (phase I) and 16/SS/0154 (phase II)), and NHS Lothian Research and Development (2016/0255). Results are disseminated through open access journals, scientific meetings, social media, newsletters anda study website (www.tebc.ed.ac.uk), and we engage with the University of Edinburgh public relations and media office to ensure maximum publicity and benefit.

Preterm Birth (PTB) accounts for approximately 11% of all births worldwide each year and is a profound physiological stressor in early life. The burden of neuropsychiatric and developmental impairment is high, with severity and prevalence correlated with gestational age at delivery. PTB is a major risk factor for the development of cerebral palsy, lower educational attainment and deficits in cognitive functioning, and individuals born preterm have higher rates of schizophrenia, autistic spectrum disorder and attention deficit/hyperactivity disorder. Factors such as gestational age at birth, systemic inflammation, respiratory morbidity, sub-optimal nutrition, and genetic vulnerability are associated with poor outcome after preterm birth, but the mechanisms linking these factors to adverse long term outcome are poorly understood. One potential mechanism linking PTB with neurodevelopmental effects is changes in the epigenome. Epigenetic processes can be defined as those leading to altered gene expression in the absence of a change in the underlying DNA sequence and include DNA methylation/hydroxymethylation and histone modifications. Such epigenetic modifications may be susceptible to environmental stimuli, and changes may persist long after the stimulus has ceased, providing a mechanism to explain the long-term consequences of acute exposures in early life. Many factors such as inflammation, fluctuating oxygenation and excitotoxicity which are known factors in PTB related brain injury, have also been implicated in epigenetic dysfunction. In this review, we will discuss the potential role of epigenetic dysregulation in mediating the effects of PTB on neurodevelopmental outcome, with specific emphasis on DNA methylation and the α-ketoglutarate dependent dioxygenase family of enzymes.

Preterm birth affects 5-18% of all babies and is associated with neurodevelopmental impairment and increased neuropsychiatric disease risk. Although preterm birth associates with differential DNA methylation at neurodevelopmental genes in buccal DNA, including leucine-rich alpha-2-glycoprotein 1 (LRG1), it is not known whether these differences also occur in the brain, or whether they persist. Thus, there is a need for animal models or in vitro systems in which to undertake longitudinal and mechanistic studies. We used a combination of in vivo rat studies and ex vivo experiments in rat cortical slices to explore their utility in modelling the human preterm brain. We identified temporal changes in DNA methylation at LRG1 in human buccal DNA over the first year of life and found persistent differences in LRG1 methylation between preterm and term infants at 1 year. These developmental changes also occurred in rat brains in vivo, alongside changes in global DNA hydroxymethylation and expression of the ten-eleven translocation (Tet1) enzyme, and were reproducible in ex vivo rat cortical slices. On the basis of the observation that neonatal glucose homeostasis can modify neurodevelopmental outcome, we studied whether glucose concentration affects Lrg1 methylation using cortical slices. Culture of slices in lower glucose concentration was associated with lower Lrg1 methylation, lower global 5hmC and Tet1 expression. Our results suggest that ex vivo organotypic cultures may be useful in the study of biological and environmental influences on the epigenome and that perturbations during early life including glucose concentration can affect methylation at specific genes implicated in neurodevelopment.

Importance: Depressive disorders arise from a combination of genetic and environmental risk factors. Epigenetic disruption provides a plausible mechanism through which gene-environment interactions lead to depression. Large-scale, epigenome-wide studies on depression are missing, hampering the identification of potentially modifiable biomarkers.Objective: To identify epigenetic mechanisms underlying depression in middle-aged and elderly persons, using DNA methylation in blood.Design, Setting, and Participants: To date, the first cross-ethnic meta-analysis of epigenome-wide association studies (EWAS) within the framework of the Cohorts for Heart and Aging Research in Genomic Epidemiology (CHARGE) Consortium was conducted. The discovery EWAS included 7948 individuals of European origin from 9 population-based cohorts. Participants who were assessed for both depressive symptoms and whole-blood DNA methylation were included in the study. Results of EWAS were pooled using sample-size weighted meta-analysis. Replication of the top epigenetic sites was performed in 3308 individuals of African American and European origin from 2 population-based cohorts.Main Outcomes and Measures: Whole-blood DNA methylation levels were assayed with Illumina-Infinium Human Methylation 450K BeadChip and depressive symptoms were assessed by questionnaire.Results: The discovery cohorts consisted of 7948 individuals (4104 [51.6%] women) with a mean (SD) age of 65.4 (5.8) years. The replication cohort consisted of 3308 individuals (2456 [74.2%] women) with a mean (SD) age of 60.3 (6.4) years. The EWAS identified methylation of 3 CpG sites to be significantly associated with increased depressive symptoms: cg04987734 (P = 1.57 × 10−08; n = 11 256; CDC42BPB gene), cg12325605 (P = 5.24 × 10−09; n = 11 256; ARHGEF3 gene), and an intergenic CpG site cg14023999 (P = 5.99 × 10−08; n = 11 256; chromosome = 15q26.1). The predicted expression of the CDC42BPB gene in the brain (basal ganglia) (effect, 0.14; P = 2.7 × 10−03) and of ARHGEF3 in fibroblasts (effect, −0.48; P = 9.8 × 10−04) was associated with major depression.Conclusions and Relevance: This study identifies 3 methylated sites associated with depressive symptoms. All 3 findings point toward axon guidance as the common disrupted pathway in depression. The findings provide new insights into the molecular mechanisms underlying the complex pathophysiology of depression. Further research is warranted to determine the utility of these findings as biomarkers of depression and evaluate any potential role in the pathophysiology of depression and their downstream clinical effects.

Impaired social function is a core feature of many psychiatric illnesses. Adverse experiences during childhood increase risk for mental illness, however it is currently unclear whether stress early in life plays a direct role in the development of social difficulties. Using an animal model of pre-pubertal stress (PPS), we investigated effects on social behaviour, oxytocin and arginine vasopressin (AVP). We also explored social performance and AVP expression in participants with borderline personality disorder (BPD) who experienced a high incidence of childhood stress. Social behaviour was impaired and AVP expression increased in animals experiencing PPS and participants with BPD. Behavioural deficits in animals were rescued through administration of the AVP receptor 1a antagonist Relcovaptan (SR49059). AVP levels and recognition of negative emotions were significantly correlated in BPD participants only. In conclusion, early life stress plays a profound role in the precipitation of social dysfunction, and AVP mediates at least part of this effect.

Non-alcoholic fatty liver disease (NAFLD) is now the commonest cause of liver disease in developed countries affecting 25–33% of the general population and up to 75% of those with obesity. Recent data suggest that alterations in DNA methylation may be related to NAFLD pathogenesis and progression and we have previously shown that dynamic changes in the cell lineage identifier 5-hydroxymethylcytosine (5hmC) may be important in the pathogenesis of liver disease. We used a model of diet-induced obesity, maintaining male mice on a high-fat diet (HFD) to generate hepatic steatosis. We profiled hepatic gene expression, global and locus-specific 5hmC and additionally investigated the effects of weight loss on the phenotype. HFD led to increased weight gain, fasting hyperglycaemia, glucose intolerance, insulin resistance and hepatic periportal macrovesicular steatosis. Diet-induced hepatic steatosis associated with reversible 5hmC changes at a discrete number of functionally important genes. We propose that 5hmC profiles are a useful signature of gene transcription and a marker of cell state in NAFLD and suggest that 5hmC profiles hold potential as a biomarker of abnormal liver physiology.

Glucocorticoids (GC) are used in paediatric practice for a broad range of conditions and all paediatricians will prescribe GC, in some form, during their career. A wide variety of GC formulations, doses and administration routes are used for periods of time ranging from days to years. Exposure to exogenous GC can result in hypothalamic-pituitary-adrenal axis suppression—otherwise known as adrenal suppression (AS). Patients with AS may be well most of the time but if GC therapy is reduced or stopped or if additional endogenous GC cannot be generated during illness, then an absolute or relative lack of GC can result in severe illness or death. Here, we highlight the relevance of AS to all paediatricians by providing an overview of the background and discussing the presentation and approaches to the management of this clinical entity.

SUMMARYBone marrow adipose tissue (BMAT) represents >10% of total adipose mass, yet unlike white or brown adipose tissues (WAT or BAT), its role in systemic metabolism remains unclear. Using transcriptomics, we reveal that BMAT is molecularly distinct to WAT but is not enriched for brown or beige adipocyte markers. Instead, pathway analysis indicated altered glucose metabolism and decreased insulin responsiveness in BMAT. We therefore tested these functions in mice and humans using positron emission tomography–computed tomography (PET/CT) with18F-fluorodeoxyglucose, including establishing a new method for BMAT identification from clinical CT scans. This revealed that BMAT resists insulin- and cold-stimulated glucose uptake and is thus functionally distinct to WAT and BAT. However, BMAT displayed greater basal glucose uptake than axial bones or subcutaneous WAT, underscoring its potential to influence systemic glucose homeostasis. These PET/CT studies are the first to characterise BMAT functionin vivoand identify BMAT as a distinct, major subtype of adipose tissue.HIGHLIGHTSBone marrow adipose tissue (BMAT) is molecularly distinct to other adipose subtypes.BMAT is less insulin responsive than WAT and, unlike BAT, is not cold-responsive.Human BMAT has greater basal glucose uptake than axial bone or subcutaneous WAT.We establish a PET/CT method for BMAT localisation and functional analysisin vivo.

Numerous studies in humans and in animal models have demonstrated that exposure to adverse environmental conditions in early life results in long-term structural and functional changes in an organism, increasing the risk of cardiometabolic, neurobehavioural and reproductive disorders in later life. Such effects are not limited to the first generation offspring but may be transmitted to a second or a number of subsequent generations, through non-genomic mechanisms. While the transmission of ‘programmed’ effects through the maternal line could occur as a consequence of multiple influences, for example, altered maternal physiology, the inheritance of effects through the male line is more difficult to explain and there is much interest in a potential role for transgenerational epigenetic inheritance. In this review, we will discuss the mechanisms by which induced effects may be transmitted through the paternal lineage, with a particular focus on the role of epigenetic inheritance. This article is part of the theme issue ‘Developing differences: early-life effects and evolutionary medicine’.

Preterm birth is a significant public health problem worldwide, leading to substantial mortality in the newborn period, and a considerable burden of complications longer term, for affected infants and their carers. The fact that it is so common, and rates vary between different populations, raising the question of whether in some circumstances it might be an adaptive trait. In this review, we outline some of the evolutionary explanations put forward for preterm birth. We specifically address the hypothesis of the predictive adaptive response, setting it in the context of the Developmental Origins of Health and Disease, and explore the predictions that this hypothesis makes for the potential causes and consequences of preterm birth. We describe how preterm birth can be triggered by a range of adverse environmental factors, including nutrition, stress and relative socioeconomic status. Examining the literature for any associated longer-term phenotypic changes, we find no strong evidence for a marked temporal shift in the reproductive life-history trajectory, but more persuasive evidence for a re-programming of the cardiovascular and endocrine system, and a range of effects on neurodevelopment. Distinguishing between preterm birth as a predictive, rather than immediate adaptive response will depend on the demonstration of a positive effect of these alterations in developmental trajectories on reproductive fitness.This article is part of the theme issue ‘Developing differences: early-life effects and evolutionary medicine'.

IntroductionGraves’ disease (Graves’ hyperthyroidism) is a challenging condition for the young person and their family. The excess thyroid hormone generated by autoimmune stimulation of the thyroid stimulating hormone receptor on the thyroid gland can have a profound impact on well-being. Managing the young person with Graves’ hyperthyroidism is more difficult than in older people because the side effects of conventional treatment are more significant in this age group and because the disease tends not to resolve spontaneously in the short to medium term. New immunomodulatory agents are available and the anti-B cell monoclonal antibody rituximab is of particular interest because it targets cells that manufacture the antibodies that stimulate the thyroid gland in Graves’.Methods and analysisThe trial aims to establish whether the combination of a single dose of rituximab (500 mg) and a 12-month course of antithyroid drug (usually carbimazole) can result in a meaningful increase in the proportion of patients in remission at 2 years, the primary endpoint. A single-stage, phase II A’Hern design is used. 27 patients aged 12–20 years with newly presenting Graves’ hyperthyroidism will be recruited. Markers of immune function, including lymphocyte numbers and antibody levels (total and specific), will be collected regularly throughout the trial.DiscussionThe trial will determine whether the immunomodulatory medication, rituximab, will facilitate remission above and beyond that observed with antithyroid drug alone. A meaningful increase in the expected proportion of young patients entering remission when managed according to the trial protocol will justify consideration of a phase III trial.Ethics and dissemination The trial has received a favourable ethical opinion (North East - Tyne and Wear South Research Ethics Committee, reference 16/NE/0253, EudraCT number 2016-000209-35). The results of this trial will be distributed at international endocrine meetings, in the peer-reviewed literature and via patient support groups.Trial registration numberISRCTN20381716.

Significance Diseases of the aging are the leading cause of morbidity and mortality. Elucidating the molecular mechanisms through which modifiable factors, such as psychosocial stress, confer risk for aging-related disease can have profound implications. By combining studies in humans with experiments in cells, we show that aging and stress synergize to epigenetically upregulate FKBP5, a protein implicated in stress physiology. Higher FKBP5 promotes inflammation by activating the master immune regulator NF-κB, whereas opposing FKBP5, either genetically or pharmacologically, prevents the effects on NF-κB. Further, the aging/stress-related epigenetic signature of FKBP5 is associated with history of myocardial infarction, a disease linked to inflammation. These findings provide molecular insights into stress-related disease, pointing to biomarker and treatment possibilities., Aging and psychosocial stress are associated with increased inflammation and disease risk, but the underlying molecular mechanisms are unclear. Because both aging and stress are also associated with lasting epigenetic changes, a plausible hypothesis is that stress along the lifespan could confer disease risk through epigenetic effects on molecules involved in inflammatory processes. Here, by combining large-scale analyses in human cohorts with experiments in cells, we report that FKBP5, a protein implicated in stress physiology, contributes to these relations. Across independent human cohorts (total n > 3,000), aging synergized with stress-related phenotypes, measured with childhood trauma and major depression questionnaires, to epigenetically up-regulate FKBP5 expression. These age/stress-related epigenetic effects were recapitulated in a cellular model of replicative senescence, whereby we exposed replicating human fibroblasts to stress (glucocorticoid) hormones. Unbiased genome-wide analyses in human blood linked higher FKBP5 mRNA with a proinflammatory profile and altered NF-κB–related gene networks. Accordingly, experiments in immune cells showed that higher FKBP5 promotes inflammation by strengthening the interactions of NF-κB regulatory kinases, whereas opposing FKBP5 either by genetic deletion (CRISPR/Cas9-mediated) or selective pharmacological inhibition prevented the effects on NF-κB. Further, the age/stress-related epigenetic signature enhanced FKBP5 response to NF-κB through a positive feedback loop and was present in individuals with a history of acute myocardial infarction, a disease state linked to peripheral inflammation. These findings suggest that aging/stress-driven FKBP5–NF-κB signaling mediates inflammation, potentially contributing to cardiovascular risk, and may thus point to novel biomarker and treatment possibilities.

Aging and psychosocial stress are associated with increased inflammation and disease risk, but the underlying molecular mechanisms are poorly understood. Because both aging and stress are also associated with lasting epigenetic changes, a plausible hypothesis is that stress exposure along the lifespan could confer disease risk by epigenetically deregulating molecules involved in inflammatory processes. Here, by combining large-scale analyses in human cohorts with mechanisticin vitroinvestigations, we found that FKBP5, a protein implicated in stress physiology, contributes to these relations. Across independent human cohorts (total n=3,131), aging and stress-related phenotypes were synergistically associated with epigenetic derepression ofFKBP5. These age/stress-related epigenetic effects were recapitulated in anin vitromodel of replicative senescence, whereby we exposed replicating human fibroblasts to stress (glucocorticoid) hormones. Unbiased genome-wide analyses in human blood linked higherFKBP5mRNA with a proinflammatory profile and altered NF-κB-related gene networks. Accordingly, experiments in immune cells showed thatFKBP5overexpression promotes inflammation by strengthening the interactions of NF-κB regulatory kinases, whereas opposing FKBP5 either by genetic deletion (CRISPR/Cas9-mediated) or selective pharmacological inhibition prevented the effects on NF-κB. Further, the age/stress-related epigenetic signature enhancedFKBP5responsivity to NF-κB through a positive feedback loop and was present in individuals with a history of acute myocardial infarction, a disease state linked to peripheral inflammation. These findings suggest that FKBP5-NF-κB signaling mediates inflammation associated with aging and stress, potentially contributing to cardiovascular risk, and may thus point to novel biomarker and treatment possibilities.SignificanceDiseases of the aging are the leading cause of morbidity and mortality. Elucidating the molecular mechanisms through which modifiable factors, such as psychosocial stress, confer risk for aging-related disease can have profound implications. Here, by combining studies in humans with experiments in cells, we find that aging and stress synergize to epigenetically derepress FKBP5, a protein implicated in stress physiology. Higher FKBP5 promotes inflammation by activating the master immune regulator NF-κB, whereas opposing FKBP5 – either genetically or pharmacologically– prevents the effects on NF-κB. Further, the age/stress-related epigenetic signature ofFKBP5is associated with history of myocardial infarction, a disease state linked to inflammation. These findings provide molecular insights into stress-related disease and may point to novel biomarker and treatment possibilities.

BackgroundPrenatal maternal obesity has been linked to adverse childhood neuropsychiatric outcomes, including increased symptoms of attention deficit hyperactivity disorder (ADHD), internalizing and externalizing problems, affective disorders and neurodevelopmental problems but few studies have studied neuropsychiatric outcomes among offspring born to very severely obese women or assessed potential familial confounding by maternal psychological distress.MethodWe evaluated neuropsychiatric symptoms in 112 children aged 3–5 years whose mothers had participated in a longitudinal study of obesity in pregnancy (50 very severe obesity, BMI ⩾40 kg/m2, obese class III and 62 lean, BMI 18.5–25 kg/m2). The mothers completed the Conners’ Hyperactivity Scale, Early Symptomatic Syndrome Eliciting Neurodevelopmental Clinical Examination Questionnaire (ESSENCE-Q), Child's Sleep Habits Questionnaire (CSHQ), Strengths and Difficulties Questionnaire (SDQ), and Child Behavior Checklist (CBCL) to assess child neuropsychiatric symptoms. Covariates included child's sex, age, birthweight, gestational age, socioeconomic deprivation levels, maternal age, parity, smoking status during pregnancy, gestational diabetes and maternal concurrent symptoms of anxiety and depression assessed using State Anxiety of Spielberger State-Trait Anxiety Index (STAI) and General Health Questionnaire (GHQ), respectively.ResultsChildren exposed to prenatal maternal very severe obesity had significantly higher scores in the Conners’ Hyperactivity Scale; ESSENCE-Q; total sleep problems in CSHQ; hyperactivity, conduct problems and total difficulties scales of the SDQ; higher externalizing and total problems, anxious/depressed, aggressive behaviour and other problem syndrome scores and higher DSM-oriented affective, anxiety and ADHD problems in CBCL. Prenatal maternal very severe obesity remained a significant predictor of child neuropsychiatric problems across multiple scales independent of demographic factors, prenatal factors and maternal concurrent symptoms of anxiety and depression.ConclusionsPrenatal maternal very severe obesity is a strong predictor of increased neuropsychiatric problems in early childhood.

Synthetic glucocorticoids are frequently used antenatally in order to reduce morbidity and mortality in babies born preterm and have been used in the management of fetuses known to be at risk of congenital adrenal hyperplasia. Although such treatment has short term advantages, evidence suggests that it can affect health in later life. Several studies have reported negative consequences of prenatal exposure to the synthetic glucocorticoid dexamethasone on offspring behavior in humans and in animal models, in association with changes in brain structure, hypothalamic–pituitary–adrenal axis function, neurotransmitter pathways, gene transcription and epigenetic regulation. These studies also highlight the importance of timing and tissue/organ-specific and sex-specific effects of prenatal glucocorticoid exposure. Here we review the evidence from human and animal studies that links prenatal synthetic glucocorticoid exposure with an increased risk for neurodevelopmental disorders.

Glucocorticoids are widely used in threatened preterm labor to promote maturation in many organ systems in preterm babies and have significant beneficial effects on morbidity and mortality. We performed transcriptional profiling in fetal liver in a rat model of prenatal glucocorticoid exposure and identified marked gene expression changes in heme biosynthesis, utilization, and degradation pathways in late gestation. These changes in gene expression associated with alterations in DNA methylation and with a reduction in hepatic heme concentration. There were no persistent differences in gene expression, DNA methylation, or heme concentrations at 4 weeks of age, suggesting that these are transient effects. Our findings are consistent with glucocorticoid-induced accelerated maturation of the haematopoietic system and support the hypothesis that glucocorticoids can drive changes in gene expression in association with alterations in DNA methylation.

STUDY QUESTION\ud \ud Do sex and maternal smoking effects on human fetal anogenital distance (AGD) persist in a larger study and how do these data integrate with the wider literature on perinatal human AGD, especially with respect to sex differences?\ud \ud \ud SUMMARY ANSWER\ud \ud Second trimester sex differences in AGD are broadly consistent with neonatal and infant measures of AGD and maternal cigarette smoking is associated with a temporary increase in male AGD in the absence of changes in circulating testosterone.\ud \ud \ud WHAT IS KNOWN ALREADY\ud \ud AGD is a biomarker of fetal androgen exposure, a reduced AGD in males being associated with cryptorchidism, hypospadias and reduced penile length. Normative fetal AGD data remain partial and windows of sensitivity of human fetal AGD to disruption are not known.\ud \ud \ud STUDY DESIGN, SIZE, DURATION\ud \ud The effects of fetal sex and maternal cigarette smoking on the second trimester (11–21 weeks of gestation) human fetal AGD were studied, along with measurement of testosterone and testicular transcripts associated with apoptosis and proliferation.\ud \ud \ud PARTICIPANTS/MATERIALS, SETTING METHODS\ud \ud AGD, measured from the centre of the anus to the posterior/caudal root of penis/clitoris (AGDapp) was determined in 56 female and 70 male morphologically normal fetuses. These data were integrated with current literature on perinatal AGD in humans.\ud \ud \ud MAIN RESULTS AND THE ROLE OF CHANCE\ud \ud At 11–13 weeks of gestation male fetal AGDapp was 61% (P< 0.001) longer than in females, increasing to 70% at 17–21 weeks. This sexual dimorphism was independent of growth characteristics (fetal weight, length, gonad weight). We confirmed that at 14–16 weeks of gestation male fetal AGDapp was increased 28% (P < 0.05) by in utero cigarette smoke exposure. Testosterone levels were not affected by smoking. To develop normative data, our findings have been integrated with available data from in vivo ultrasound scans and neonatal studies. Inter-study variations in male/female AGD differences lead to the conclusion that normalization and standardization approaches should be developed to enable confidence in comparing data from different perinatal AGD studies.\ud \ud \ud LIMITATIONS, REASONS FOR CAUTION\ud \ud Sex differences, and a smoking-dependent increase in male fetal AGD at 14–16 weeks, identified in a preliminary study, were confirmed with a larger number of fetuses. However, human fetal AGD should, be re-assessed once much larger numbers of fetuses have been studied and this should be integrated with more detailed analysis of maternal lifestyle. Direct study of human fetal genital tissues is required for further mechanistic insights.\ud \ud \ud WIDER IMPLICATIONS OF THE FINDINGS\ud \ud Fetal exposure to cigarette smoke chemicals is known to lead to reduced fertility in men and women. Integration of our data into the perinatal human AGD literature shows that more work needs to be done to enable reliable inter-study comparisons.\ud \ud \ud STUDY FUNDING/COMPETING INTEREST(S)\ud \ud The study was supported by grants from the Chief Scientist Office (Scottish Executive, CZG/1/109 & CZG/4/742), NHS Grampian Endowments (08/02), the European Community's Seventh Framework Programme (FP7/2007-2013) under grant agreement no 212885 and the Medical Research Council, UK (MR/L010011/1). The authors declare they have no competing interests, be it financial, personal or professional.

A regular subject of discussion on neonatal ward rounds is the rate of weight gain in preterm infants. This is because we believe that good nutrition is important for optimal body growth, including brain growth and development. In this issue, Andrews et al 1 demonstrate that it is possible to get close to what many consider the holy grail of preterm nutrition, which is matching the rate of fetal growth as displayed on the WHO preterm growth chart. In this article, we identify some of the difficulties with this concept and suggest priorities for research. The third trimester of pregnancy is a period of rapid growth. Normal fetal physiology is abruptly challenged at the time of preterm delivery, by the difficulty in sustaining the rate of nutrient supply, and as a result of major changes in environment including: (1) air breathing and increased tissue oxygen availability; (2) milk feeding; and (3) bacterial colonisation. The physiological alterations, including major changes in gene expression resulting from adaptation to these new conditions, mean that the use of fetal growth as a standard for preterm infants, and assumptions about weight gain as a surrogate for the quality of neonatal tissue growth need to be considered carefully. Very low birthweight preterm infants frequently develop an early fall-off in neonatal weight centile level (‘centile lag’) when assessed against a fetal growth standard and tend to occupy a lower weight centile at discharge than at birth.2 This is accompanied by a nutrient deficit when compared with the same period in fetal life.3 Much has been made of the correlation between a greater centile lag in the neonatal period and neurodevelopmental impairment4 despite the likelihood of residual confounding factors and the lack of data from intervention trials. This has encouraged the assumption that the greater the weight …