Differential DNA methylation of steatosis and non-alcoholic fatty liver disease in adolescence.

BACKGROUND AND AIMS: Epigenetic modifications are associated with hepatic fat accumulation and non-alcoholic fatty liver disease (NAFLD). However, few epigenetic modifications directly implicated in such processes have been identified during adolescence, a critical developmental window where physiological changes could influence future disease trajectory. To investigate the association between DNA methylation and NAFLD in adolescence, we undertook discovery and validation of novel methylation marks, alongside replication of previously reported marks. APPROACH AND RESULTS: We performed a DNA methylation epigenome-wide association study (EWAS) on DNA from whole blood from 707 Raine Study adolescents phenotyped for steatosis score and NAFLD by ultrasound at age 17. Next, we performed pyrosequencing validation of loci within the most 100 strongly associated differentially methylated CpG sites (dmCpGs) for which ≥ 2 probes per gene remained significant across four statistical models with a nominal p value < 0.007. EWAS identified dmCpGs related to three genes (ANK1, MIR10a, PTPRN2) that met our criteria for pyrosequencing. Of the dmCpGs and surrounding loci that were pyrosequenced (ANK1 n = 6, MIR10a n = 7, PTPRN2 n = 3), three dmCpGs in ANK1 and two in MIR10a were significantly associated with NAFLD in adolescence. After adjustment for waist circumference only dmCpGs in ANK1 remained significant. These ANK1 CpGs were also associated with γ-glutamyl transferase and alanine aminotransferase concentrations. Three of twenty-two differentially methylated dmCpGs previously associated with adult NAFLD were associated with NAFLD in adolescence (all adjusted p < 2.3 × 10-3). CONCLUSIONS: We identified novel DNA methylation loci associated with NAFLD and serum liver biochemistry markers during adolescence, implicating putative dmCpG/gene regulatory pathways and providing insights for future mechanistic studies.

Adiposity associated DNA methylation signatures in adolescents are related to leptin and perinatal factors.

Epigenetics links perinatal influences with later obesity. We identifed differentially methylated CpG (dmCpG) loci measured at 17 years associated with concurrent adiposity measures and examined whether these were associated with hsCRP, adipokines, and early life environmental factors. Genome-wide DNA methylation from 1192 Raine Study participants at 17 years, identified 29 dmCpGs (Bonferroni corrected p < 1.06E-07) associated with body mass index (BMI), 10 with waist circumference (WC) and 9 with subcutaneous fat thickness. DmCpGs within Ras Association (RalGDS/AF-6), Pleckstrin Homology Domains 1 (RAPH1), Musashi RNA-Binding Protein 2 (MSI2), and solute carrier family 25 member 10 (SLC25A10) are associated with both BMI and WC. Validation by pyrosequencing confirmed these associations and showed that MSI2 , SLC25A10 , and RAPH1 methylation was positively associated with serum leptin. These were  also associated with the early environment; MSI2 methylation (ß = 0.81, p = 0.0004) was associated with pregnancy maternal smoking, SLC25A10 (CpG2 ß = 0.12, p = 0.002) with pre- and early pregnancy BMI, and RAPH1 (ß = -1.49, p = 0.036) with gestational weight gain. Adjusting for perinatal factors, methylation of the dmCpGs within MSI2, RAPH1, and SLC25A10 independently predicted BMI, accounting for 24% of variance. MSI2 methylation was additionally associated with BMI over time (17 years old ß = 0.026, p = 0.0025; 20 years old ß = 0.027, p = 0.0029) and between generations (mother ß = 0.044, p = 7.5e-04). Overall findings suggest that DNA methylation in MSI2, RAPH1, and SLC25A10 in blood may be robust markers, mediating through early life factors.

Utilising surface-level data to explore surface, tooth, individual and family influence on the aetiology of hypomineralised second primary molars.

OBJECTIVES: Hypomineralised second primary molars (HSPM) are common developmental enamel defects. The aims of this study were to use surface-level data to explore the clustering of HSPM at four levels (family, child, tooth, surface). METHODS: This study of 172 twin pairs was nested within the Peri/postnatal Epigenetic Twin Study. HSPM was measured by standardised oral examinations at age 6 years. Multilevel logistic regression models were fitted to assess the correlation structure of surface level data and variation in HSPM. The associations between surface level risk factors and HSPM were then explored using the multilevel logistic regression model using the best fitting correlation structure. RESULTS: The prevalence of HSPM was 68 (19.8%) children, with a total of 141 (10.3%) teeth and 264 tooth surfaces (6.3%) affected. Multilevel models revealed that a hierarchical structure accounting for correlation at the family, child and tooth level best accounted for the variation in HSPM. The estimated variances from the best fitting model (Model 3) were largest at the family level (12.27, 95% CI 6.68, 22.51) compared with 5.23 at the child level and 1.93 at the tooth level. Application of regression analysis utilising this three-level correlation structure identified tooth/surface level factors in addition to the previously identified familial and individual risk factors for HSPM. CONCLUSION: In addition to familial (environmental and genetic) and unique child-level factors, the aetiology of HSPM is likely to be influenced by local tooth-level factors.

Additives in pet food: are they safe?

A good, nutritious diet is essential for the health and well-being of our domestic pets. Today, most pet dogs and cats are fed highly processed food bearing little resemblance to canine and feline ancestral diets. Additives are included in processed pet food to provide nutritional benefits, ensure food safety, and maintain the desirable features of colour, flavour, texture, stability and resistance to spoilage. This paper reviews the safety of various additives in processed pet food. Labelling, safety assessment, and ethical concerns regarding existing toxicity testing procedures are also considered. The adequacy of testing for many additives and the scientific basis for determining safety are questioned. Additives can be synthetic or 'natural' although the distinction can be blurred when naturally derived substances are synthesised in the laboratory, or extracted using a high level of physical and chemical processing. Although additives play important roles in processed food production, updated strategies and technologies may be required to establish their safety in the pet food industry.

A twin study of body mass index and dental caries in childhood.

Sub-optimal nutrition and dental caries are both common with significant short and long-term implications for child health and development. We applied twin statistical methods to explore the relationship between body mass index (BMI) and dental caries. We measured BMI at 18 months and six years of age and cumulative dental caries experience at six years in 344 twin children. Dental caries in primary teeth was categorised into 'any' or 'advanced' and BMI was analysed as both a continuous and categorical variable. Statistical analyses included multiple logistic regression using generalized estimating equations and within/between-pair analyses. There was no association between BMI and 'any' dental caries experience at either time-point, neither overall nor in within/between pair analyses. However, 'advanced' dental caries at six years was associated with a within-pair difference in BMI of -0.55 kg/m2 (95% CI -1.00, -0.11, p = 0.015). A within-pair increase of 1 kg/m2 in BMI was associated with a lower within-pair risk of advanced dental caries (OR 0.68, 95% CI 0.52, 0.90, p = 0.007). These findings reveal a possible causal relationship between lower BMI and dental caries. As dental outcomes were only measured at one time point, the direction of this potentially causal relationship is unclear.

The CODATwins Project: The Current Status and Recent Findings of COllaborative Project of Development of Anthropometrical Measures in Twins.

The COllaborative project of Development of Anthropometrical measures in Twins (CODATwins) project is a large international collaborative effort to analyze individual-level phenotype data from twins in multiple cohorts from different environments. The main objective is to study factors that modify genetic and environmental variation of height, body mass index (BMI, kg/m2) and size at birth, and additionally to address other research questions such as long-term consequences of birth size. The project started in 2013 and is open to all twin projects in the world having height and weight measures on twins with information on zygosity. Thus far, 54 twin projects from 24 countries have provided individual-level data. The CODATwins database includes 489,981 twin individuals (228,635 complete twin pairs). Since many twin cohorts have collected longitudinal data, there is a total of 1,049,785 height and weight observations. For many cohorts, we also have information on birth weight and length, own smoking behavior and own or parental education. We found that the heritability estimates of height and BMI systematically changed from infancy to old age. Remarkably, only minor differences in the heritability estimates were found across cultural-geographic regions, measurement time and birth cohort for height and BMI. In addition to genetic epidemiological studies, we looked at associations of height and BMI with education, birth weight and smoking status. Within-family analyses examined differences within same-sex and opposite-sex dizygotic twins in birth size and later development. The CODATwins project demonstrates the feasibility and value of international collaboration to address gene-by-exposure interactions that require large sample sizes and address the effects of different exposures across time, geographical regions and socioeconomic status.

Food intolerance in dogs and cats.

Food intolerance refers to any abnormal physiological response to a food or food additive believed not to be immunological in nature. Mechanisms include food toxicity, pharmacological reactions, metabolic reactions, dysmotility, dysbiosis, physical effects and non-specific dietary sensitivity. Food intolerance reactions are variable, typically dose-dependent, and can occur at any age. Signs may arise at any time, sometimes several hours or days after consumption of the offending food item, and can last for hours or days. Dietary indiscretion and non-immunological food intolerance are probably more common in dogs than true dietary hypersensitivity. Hopefully, with a greater knowledge of the different pathophysiological mechanisms involved, we will become better at recognising, preventing and managing adverse food reactions.

Etiology of Hypomineralized Second Primary Molars: A Prospective Twin Study.

The etiology of hypomineralized second primary molars (HSPM) is unclear, but genetic and environmental factors have been proposed. The aim of this study was to investigate the relative contribution of genes and environment to the etiology of HSPM and to identify potential environmental risk factors in a longitudinal twin cohort. Children from twin pregnancies ( N = 250) were recruited antenatally, and detailed demographic, health, and phenotypic data were collected at recruitment, 24- and 36-wk gestation, birth, and 18 mo of age. 25-Hydroxyvitamin D was quantified for mothers at 28-wk gestation and infants at birth. Dental examinations were conducted on the twins at 6 y of age to determine the presence, severity, and extent of HSPM per standardized criteria. To investigate associations of environmental risk factors with HSPM, multiple logistic regression models were fitted with generalized estimating equations to adjust for twin correlation. Within- and between-pair analyses were performed for unshared continuous variables: birthweight and birth 25-hydroxyvitamin D. Twin-twin concordance for monozygotic (MZ) and dizygotic (DZ) pairs was calculated and compared after adjusting for identified risk factors. A total of 344 twins underwent the 6-y-old dental assessment; HSPM occurred in 68 (19.8%). After adjusting for potential confounders, vitamin D levels at birth, infantile eczema, dizygosity, in vitro fertilization, socioeconomic position, and maternal smoking beyond the first trimester of pregnancy demonstrated the strongest associations with HSPM. Overall concordance for HSPM was 0.47 (95% CI, 0.32 to 0.62) with weak evidence ( P = 0.078) of higher concordance in MZ twins (0.63; 95% CI, 0.38 to 0.89) as compared with DZ twins (0.41; 95% CI, 0.24 to 0.58). After adjusting for known risk factors, there was no evidence ( P = 0.172) for an additive genetic influence. These findings suggest that shared and unshared environmental factors, such as maternal smoking later in pregnancy and infantile eczema, are important in the etiology of HSPM.

Epigenetics and DOHaD: from basics to birth and beyond.

Developmental origins of health and disease (DOHaD) is the study of how the early life environment can impact the risk of chronic diseases from childhood to adulthood and the mechanisms involved. Epigenetic modifications such as DNA methylation, histone modifications and non-coding RNAs are involved in mediating how early life environment impacts later health. This review is a summary of the Epigenetics and DOHaD workshop held at the 2016 DOHaD Society of Australia and New Zealand Conference. Our extensive knowledge of how the early life environment impacts later risk for chronic disease would not have been possible without animal models. In this review we highlight some animal model examples that demonstrate how an adverse early life exposure results in epigenetic and gene expression changes that may contribute to increased risk of chronic disease later in life. Type 2 diabetes and cardiovascular disease are chronic diseases with an increasing incidence due to the increased number of children and adults that are obese. Epigenetic changes such as DNA methylation have been shown to be associated with metabolic health measures and potentially predict future metabolic health status. Although more difficult to elucidate in humans, recent studies suggest that DNA methylation may be one of the epigenetic mechanisms that mediates the effects of early life exposures on later life risk of obesity and obesity related diseases. Finally, we discuss the role of the microbiome and how it is a new player in developmental programming and mediating early life exposures on later risk of chronic disease.

Antecedent Nippostrongylus infection alters the lung immune response to Plasmodium berghei.

In endemic regions, it is not uncommon for patients to be co-infected with soil-transmitted helminths and malaria. Although both malaria and many helminth species use the lungs as a site of development, little attention has been paid to the impact that pulmonary immunity induced by one parasite has on the lung response to the other. To model the consequences of a prior hookworm exposure on the development of immunity to malaria in the lungs, mice were infected with Nippostrongylus brasiliensis and 2 weeks later challenged with Plasmodium berghei. We found that a pre-existing hookworm-induced type 2 immune environment had a measurable but modest impact on the nature of the malaria-driven type 1 cytokine response in the lungs that was associated with a transient effect on parasite development and no significant changes in morbidity and mortality after malaria infection. However, prior hookworm infection did have a lasting effect on lung macrophages, where the malaria-induced M1-like response was blunted by previous M2 polarization. These results demonstrate that, although helminth parasites confer robust changes to the immunological status of the pulmonary microenvironment, lung immunity is plastic and capable of rapidly adapting to consecutive heterologous infections.

mTORC2 signalling regulates M2 macrophage differentiation in response to helminth infection and adaptive thermogenesis.

Alternatively activated macrophages (M2) have an important function in innate immune responses to parasitic helminths, and emerging evidence also indicates these cells are regulators of systemic metabolism. Here we show a critical role for mTORC2 signalling in the generation of M2 macrophages. Abrogation of mTORC2 signalling in macrophages by selective conditional deletion of the adaptor molecule Rictor inhibits the generation of M2 macrophages while leaving the generation of classically activated macrophages (M1) intact. Selective deletion of Rictor in macrophages prevents M2 differentiation and clearance of a parasitic helminth infection in mice, and also abrogates the ability of mice to regulate brown fat and maintain core body temperature. Our findings define a role for mTORC2 in macrophages in integrating signals from the immune microenvironment to promote innate type 2 immunity, and also to integrate systemic metabolic and thermogenic responses.

The establishment of DOHaD working groups in Australia and New Zealand.

The evidence underpinning the developmental origins of health and disease (DOHaD) is overwhelming. As the emphasis shifts more towards interventions and the translational strategies for disease prevention, it is important to capitalize on collaboration and knowledge sharing to maximize opportunities for discovery and replication. DOHaD meetings are facilitating this interaction. However, strategies to perpetuate focussed discussions and collaborations around and between conferences are more likely to facilitate the development of DOHaD research. For this reason, the DOHaD Society of Australia and New Zealand (DOHaD ANZ) has initiated themed Working Groups, which convened at the 2014-2015 conferences. This report introduces the DOHaD ANZ Working Groups and summarizes their plans and activities. One of the first Working Groups to form was the ActEarly birth cohort group, which is moving towards more translational goals. Reflecting growing emphasis on the impact of early life biodiversity - even before birth - we also have a Working Group titled Infection, inflammation and the microbiome. We have several Working Groups exploring other major non-cancerous disease outcomes over the lifespan, including Brain, behaviour and development and Obesity, cardiovascular and metabolic health. The Epigenetics and Animal Models Working Groups cut across all these areas and seeks to ensure interaction between researchers. Finally, we have a group focussed on 'Translation, policy and communication' which focusses on how we can best take the evidence we produce into the community to effect change. By coordinating and perpetuating DOHaD discussions in this way we aim to enhance DOHaD research in our region.

Enhanced resolution of experimental ARDS through IL-4-mediated lung macrophage reprogramming.

Despite intense investigation, acute respiratory distress syndrome (ARDS) remains an enormous clinical problem for which no specific therapies currently exist. In this study, we used intratracheal lipopolysaccharide or Pseudomonas bacteria administration to model experimental acute lung injury (ALI) and to further understand mediators of the resolution phase of ARDS. Recent work demonstrates macrophages transition from a predominant proinflammatory M1 phenotype during acute inflammation to an anti-inflammatory M2 phenotype with ALI resolution. We tested the hypothesis that IL-4, a potent inducer of M2-specific protein expression, would accelerate ALI resolution and lung repair through reprogramming of endogenous inflammatory macrophages. In fact, IL-4 treatment was found to offer dramatic benefits following delayed administration to mice subjected to experimental ALI, including increased survival, accelerated resolution of lung injury, and improved lung function. Expression of the M2 proteins Arg1, FIZZ1, and Ym1 was increased in lung tissues following IL-4 treatment, and among macrophages, FIZZ1 was most prominently upregulated in the interstitial subpopulation. A similar trend was observed for the expression of macrophage mannose receptor (MMR) and Dectin-1 on the surface of alveolar macrophages following IL-4 administration. Macrophage depletion or STAT6 deficiency abrogated the therapeutic effect of IL-4. Collectively, these data demonstrate that IL-4-mediated therapeutic macrophage reprogramming can accelerate resolution and lung repair despite delayed use following experimental ALI. IL-4 or other therapies that target late-phase, proresolution pathways may hold promise for the treatment of human ARDS.

Association of in vitro fertilization with global and IGF2/H19 methylation variation in newborn twins.

In vitro fertilization (IVF) and its subset intracytoplasmic sperm injection (ICSI), are widely used medical treatments for conception. There has been controversy over whether IVF is associated with adverse short- and long-term health outcomes of offspring. As with other prenatal factors, epigenetic change is thought to be a molecular mediator of any in utero programming effects. Most studies focused on DNA methylation at gene-specific and genomic level, with only a few on associations between DNA methylation and IVF. Using buccal epithelium from 208 twin pairs from the Peri/Postnatal Epigenetic Twin Study (PETS), we investigated associations between IVF and DNA methylation on a global level, using the proxies of Alu and LINE-1 interspersed repeats in addition to two locus-specific regulatory regions within IGF2/H19, controlling for 13 potentially confounding factors. Using multiple correction testing, we found strong evidence that IVF-conceived twins have lower DNA methylation in Alu, and weak evidence of lower methylation in one of the two IGF2/H19 regulatory regions and LINE-1, compared with naturally conceived twins. Weak evidence of a relationship between ICSI and DNA methylation within IGF2/H19 regulatory region was found, suggesting that one or more of the processes associated with IVF/ICSI may contribute to these methylation differences. Lower within- and between-pair DNA methylation variation was also found in IVF-conceived twins for LINE-1, Alu and one IGF2/H19 regulatory region. Although larger sample sizes are needed, our results provide additional insight to the possible influence of IVF and ICSI on DNA methylation. To our knowledge, this is the largest study to date investigating the association of IVF and DNA methylation.

Helminths in the lungs.

Parasitic helminths infect well over one billion people and typically cause chronic and recurrent infections that exert a considerable toll on human health and productivity. A significant number of important intestinal- and tissue-dwelling helminth parasites have evolved a scripted migration through select organ systems. Of specific interest here are the helminth parasites that interact with respiratory tissues and the pulmonary immune system. This review will consider the nature of the interactions between helminth parasites and the lung environment, as well as the consequences of these interactions on the evolution of parasitism and host immunity.

Elastase-coupled beads as a tool for characterizing localized alveolar tissue destruction associated with the onset of emphysema.

Intratracheal elastase challenge of laboratory animals has long been established as a model for observing the physiological and morphological changes that result from alveolar destruction, the hallmark of emphysema. However, instillation of elastase suspended in buffer results in widespread inflammation and variable emphysematous lesions, which has made the identification of specific cellular and molecular events associated with the onset of emphysema difficult to define. Here we establish a bead-based elastase delivery system that induces localized tissue destruction, a key event in the initiation of emphysema. Elastase was coupled to bisacrylamide beads, which were shown to retain enzymatic activity prior to intratracheal administration in mice. C57BL/6 mice were given a single dose of 40,000 beads, which became distributed throughout the small airways and parenchyma of the lung. Elastase-coupled beads resulted in a quantifiable loss of alveolar tissue immediately surrounding the beads, an effect that was not observed with beads that lacked protein altogether or with beads containing elastase inactivated by an irreversible inhibitor. Furthermore, beads bound with active elastase elicited local recruitment of mononuclear cells, including macrophages, and polymorphonuclear neutrophils to the site of bead deposition, a feature consistent with the cellular infiltration observed following conventional solubilized elastase challenges. This work identifies a novel bead-based enzyme delivery system that also extends the elastase model of emphysema to permit the characterization of mechanisms that drive alveolar surface area loss following elastin degradation in focal emphysematous lesions.

Decreased cortical muscarinic M1 receptors in schizophrenia are associated with changes in gene promoter methylation, mRNA and gene targeting microRNA.

Many studies have shown decreased cortical muscarinic M1 receptors (CHRM1) in schizophrenia (Sz), with one study showing Sz can be separated into two populations based on a marked loss of CHRM1 (-75%) in -25% of people (Def-Sz) with the disorder. To better understand the mechanism contributing to the loss of CHRM1 in Def-Sz, we measured specific markers of gene expression in the cortex of people with Sz as a whole, people differentiated into Def-Sz and people with Sz that do not have a deficit in cortical CHRM1 (Non-Def-Sz) and health controls. We now report that cortical CHRM1 gene promoter methylation and CHRM1 mRNA are decrease in Sz, Def-Sz and Non-Def-Sz but levels of the micro RNA (miR)-107, a CHRM1 targeting miR, are increased only in Def-Sz. We also report in vitro data strongly supporting the notion that miR-107 levels regulate CHRM1 expression. These data suggest there is a reversal of the expected inverse relationship between gene promoter methylation and CHRM1 mRNA in people with Sz and that a breakdown in gene promoter methylation control of CHRM1 expression is contributing to the global pathophysiology of the syndrome. In addition, our data argues that increased levels of at least one miR, miR-107, is contributing to the marked loss of cortical CHRM1 in Def-Sz and this may be a differentiating pathophysiology. These latter data continue to support the hypothesis that microRNAs (miRNA) have a role in the underlying neurobiology of Sz but argue they are differentially affected in subsets of people within that syndrome.

Distinct patterns of gene-specific methylation in mammalian placentas: implications for placental evolution and function.

The placenta has arisen relatively recently and is among the most rapidly evolving tissues in mammals. Several different placental barrier and structure types appear to have independently evolved common functional features. Specific patterns of gene expression that determine placental development in humans are predicted to be accompanied by specific profiles of epigenetic modification. However, the stratification of epigenetic modifications into those involved in conserved aspects of placental function, versus those involved in divergent placental features, has yet to begin. As a first step towards this goal, we have investigated the methylation status of a small number of gene-specific methylation events recently identified in human placenta, in a panel of placental tissue from baboon, marmoset, cow, cat, guinea pig and mouse. These represent disparate placental barrier types and structures. In this study we hypothesized that specific epigenetic markings may be associated with placental barrier type or function, independent of phylogeny. However, in contrast to our predictions, the majority of gene-specific methylation appears to track with phylogeny, independent of placental barrier type or other structural features. This suggests that despite the likelihood of epigenetic modification playing a role in the functioning and evolution of different placental subtypes, there is no evidence for an involvement of the gene-specific methylation profiles we have identified, in specifying these differences. Further studies, examining larger numbers of epigenetic modifications across phylogeny, are required to define the role of specific epigenetic modifications in the evolution of distinct placental structures.