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.

DNA methylation patterns within whole blood of adolescents born from assisted reproductive technology are not different from adolescents born from natural conception.

STUDY QUESTION: Do the epigenome-wide DNA methylation profiles of adolescents born from ART differ from the epigenome of naturally conceived counterparts? SUMMARY ANSWER: No significant differences in the DNA methylation profiles of adolescents born from ART [IVF or ICSI] were observed when compared to their naturally conceived, similar aged counterparts. WHAT IS KNOWN ALREADY: Short-term and longer-term studies have investigated the general health outcomes of children born from IVF treatment, albeit without common agreement as to the cause and underlying mechanisms of these adverse health findings. Growing evidence suggests that the reported adverse health outcomes in IVF-born offspring might have underlying epigenetic mechanisms. STUDY DESIGN, SIZE, DURATION: The Growing Up Healthy Study (GUHS) is a prospective study that recruited 303 adolescents and young adults, conceived through ART, to compare various long-term health outcomes and DNA methylation profiles with similar aged counterparts from Generation 2 from the Raine Study. GUHS assessments were conducted between 2013 and 2017. The effect of ART on DNA methylation levels of 231 adolescents mean age 15.96 ± 1.59 years (52.8% male) was compared to 1188 naturally conceived counterparts, 17.25 ± 0.58 years (50.9% male) from the Raine Study. PARTICIPANTS/MATERIALS, SETTING, METHODS: DNA methylation profiles from a subset of 231 adolescents (13-19.9 years) from the GUHS, generated using the Infinium Methylation Epic Bead Chip (EPIC) array were compared to 1188 profiles from the Raine Study previously measured using the Illumina 450K array. We conducted epigenome-wide association approach (EWAS) and tested for an association between the cohorts applying Firth's bias reduced logistic regression against the outcome of ART versus naturally conceived offspring. Additionally, within the GUHS cohort, we investigated differences in methylation status in fresh versus frozen embryo transfers, cause of infertility as well as IVF versus ICSI conceived offspring. Following the EWAS analysis we investigated nominally significant probes using Gene Set Enrichment Analysis (GSEA) to identify enriched biological pathways. Finally, within GUHS we compared four estimates (Horvath, Hanuum, PhenoAge [Levine], and skin Horvath) of epigenetic age and their correlation with chronological age. MAIN RESULTS AND THE ROLE OF CHANCE: Between the two cohorts, we did not identify any DNA methylation probes that reached a Bonferroni corrected P-value < 1.24E-0.7. When comparing IVF versus ICSI conceived adolescents within the GUHS cohort, after adjustment for participant age, sex, maternal smoking, multiple births, and batch effect, three methylation probes (cg15016734, cg26744878 and cg20233073) reached a Bonferroni correction of 6.31E-08. After correcting for cell count heterogeneity, two of the aforementioned probes remained significant and an additional two probes (cg 0331628 and cg 20235051) were identified. A general trend towards hypomethylation in the ICSI offspring was observed. All four measures of epigenetic age were highly correlated with chronological age and showed no evidence of accelerated epigenetic aging within their whole blood. LIMITATIONS, REASONS FOR CAUTION: The small sample size coupled with the use of whole blood, where epigenetic differences may occur in other tissue. This was corrected by the utilized statistical method that accounts for imbalanced sample size between groups and adjusting for cell count heterogeneity. Only a small portion of the methylome was analysed and rare individual differences may be missed. WIDER IMPLICATIONS OF THE FINDINGS: Our findings provide further reassurance that the effects of the ART manipulations occurring during early embryogenesis, existing in the neonatal period are indeed of a transient nature and do not persist into adolescence. However, we have not excluded that alternative epigenetic mechanisms may be at play. STUDY FUNDING/COMPETING INTEREST(S): This project was supported by NHMRC project Grant no. 1042269 and R.J.H. received funding support from Ferring Pharmaceuticals Pty Ltd. R.J.H. is the Medical Director of Fertility Specialists of Western Australia and a shareholder in Western IVF. He has received educational sponsorship from Merck Sharp & Dohme Corp.- Australia, Merck-Serono Australia Pty Ltd and Ferring Pharmaceuticals Pty Ltd. P.B. is the Scientific Director of Concept Fertility Centre, Subiaco, Western Australia. J.L.Y. is the Medical Director of PIVET Medical Centre, Perth, Western Australia. The remaining authors have no conflicts of interest.

Machine learning and clinical epigenetics: a review of challenges for diagnosis and classification.

BACKGROUND: Machine learning is a sub-field of artificial intelligence, which utilises large data sets to make predictions for future events. Although most algorithms used in machine learning were developed as far back as the 1950s, the advent of big data in combination with dramatically increased computing power has spurred renewed interest in this technology over the last two decades. MAIN BODY: Within the medical field, machine learning is promising in the development of assistive clinical tools for detection of e.g. cancers and prediction of disease. Recent advances in deep learning technologies, a sub-discipline of machine learning that requires less user input but more data and processing power, has provided even greater promise in assisting physicians to achieve accurate diagnoses. Within the fields of genetics and its sub-field epigenetics, both prime examples of complex data, machine learning methods are on the rise, as the field of personalised medicine is aiming for treatment of the individual based on their genetic and epigenetic profiles. CONCLUSION: We now have an ever-growing number of reported epigenetic alterations in disease, and this offers a chance to increase sensitivity and specificity of future diagnostics and therapies. Currently, there are limited studies using machine learning applied to epigenetics. They pertain to a wide variety of disease states and have used mostly supervised machine learning methods.

Patterns of DNA Methylation Across the Leptin Core Promoter in Four Diverse Asian and North American Populations.

DNA methylation is the most widely studied of epigenetic mechanisms, with environmental effects recorded through patterned attachments of methyl groups along the DNA that are capable of modifying gene expression without altering the DNA sequencing. The degree to which these patterns of DNA methylation are heritable, the expected range of normality across populations, and the phenotypic relevance of pattern variation remain unclear. Genes regulating metabolic pathways appear to be vulnerable to ongoing nutritional programming over the life course, as dietary nutrients are significant environmental determinants of DNA methylation, supplying both the methyl groups and energy to generate the methylation process. Here we examine methylation patterns along a region of the metabolic gene leptin (LEP). LEP's putative functions include regulation of energy homeostasis, with its signals affecting energy intake and expenditure, adipogenesis and energy storage, lipid and glucose metabolism, bone metabolism, and reproductive endocrine function. A pattern of differential methylation across CpG sites of the LEP core promoter has been previously identified; however, any consistency of pattern or its phenotypic significance is not fully elucidated among populations. Using DNA extracted from unfractionated white blood cells of peripheral blood samples, our pilot study, divided into two parts, examined the significance of variation in DNA methylation patterns along the leptin core promoter in four populations (phase 1) and used biomarkers reflecting leptin's functional process in two of those populations, western Buryat of Siberia and the Mennonite of central Kansas, to investigate the relevance of the ethnic variation identified in the DNA methylation (phase 2). LEP's core promoter region contains both the binding site for C/EBPα (CCAAT/enhancer binding protein alpha), which tempers the final step in adipocyte maturity and capacity to synthesize leptin, and the TATA motif controlling leptin synthesis. Previous studies report that increased methylation in this region is correlated to decreased gene expression, suggesting tissue-specific methylation variation at this region ( Melzner et al. 2002 ). We hypothesized that evidence of nutritional epigenetic programming would be identified through variation in patterns of DNA methylation and that functional relevance of that variation among populations would be identified through biomarkers that reflect leptin's metabolic signals: serum leptin levels, lipoproteins of the lipid transport system, and anthropometric measures. In phase 1, our combined analyses of 313 individuals documented a distinct and consistent overall pattern of differential DNA methylation across seven CpG sites of LEP core promoter in all ethnicities and both sexes. This pattern replicates those identified in previous studies, suggesting a conserved core promoter region across populations. Phase 2 analyses of two of the four populations (n = 239), correlating methylation at the C/EBPα transcription binding site (TBS) with metabolic and anthropometric biomarkers reflecting LEP roles, showed that stature, which reflects bone growth and remodeling, was significantly and inversely correlated with the percentage of DNA methylation at this site in both sexes. We suggest that variation in DNA methylation along the LEP core promoter plays a substantial role in energy signals affecting both adipogenesis and bone metabolism.

OS070. Shared genetic risk factors for preeclampsia and cardiovascular disease.

INTRODUCTION: There is compelling evidence to support the hypothesis that a maternal constitutional predisposition to cardiovascular disease (CVD) is a key component in development of preeclampsia. In particular, CVD and preeclampsia share pathological features such as endothelial dysfunction and inflammation, and have several metabolic abnormalities in common. In support of this hypothesis, our recent genetic dissection of the Australian preeclampsia susceptibility locus on chromosome 2q22 revealed shared novel genetic risk factors for preeclampsia and CVD-related traits. OBJECTIVES: To replicate association between our recently reported 2q22 preeclampsia risk variants and CVD-related traits in an independent Australian population based cohort. METHODS: Four independent SNPs from four genes, rs35821928 (LRP1B), rs17783344 (GCA), rs115015150 (RND3) and rs2322659 (LCT), were recently found to be significantly associated with preeclampsia susceptibility and CVD-related traits. These SNPs were genotyped in a large independent Australian cohort rich in quantitative CVD risk traits; The Western Australian Pregnancy Cohort (Raine) Study. This cohort comprises of blood samples from 1246 mothers and 1461 adolescents and clinical measures such as, but not limited to, anthropometric measures of adiposity and lipid-related measures. Genetic association analyses of these four potential preeclampsia susceptibility SNPs against the CVD-related risk traits were performed using the software package R. All statistical analyses assumed an additive model of gene action. RESULTS: Several significant associations (p<0.05) for all four SNPs with a variety of CVD-related risk traits were detected, both for the mothers and the adolescents. The LRP1B SNP was associated with HDL/cholesterol ratio, LDL cholesterol, triglycerides, skinfold measures and weight. The GCA SNP was associated with total cholesterol, HDL cholesterol, serum insulin, hemoglobin, blood glucose, BMI and skinfold measures. The RND3 SNP was associated with triglycerides and waist-hip ratio. The LCT SNP was associated with hemoglobin, blood glucose and abdominal skinfold. CONCLUSION: We have recently identified genetic variants within the LRP1B, GCA, RND3 and LCT genes to be significantly associated with preeclampsia susceptibility and CVD-related risk traits. We have now demonstrated thatthese specific genetic variants are associated with CVD-related risk traits in an independent population. Our collective findings provide substantial empirical data to support the hypothesis that genetic risk factors for preeclampsia and CVD are, at least in part, shared.

[Comparative analysis of mitochondrial DNA of Yakuts and other Asian populations].

Mitochondrial DNA of Yakuts has been compared to those of other Asian populations that belong to the Turkic, Mongolic, and Manchu-Tungusic linguistic groups. Haplogroups C and D proved to be the most frequent ones in Yakuts. In contrast to other Asian populations, subcluster D5a is major in Yakuts. The results have demonstrated that Yakuts are close to Tuvinians and Altaians in maternal lineage.

Violence prevention: myth or reality?

Newspapers and television daily attest to the fact that violence is a pervasive element in our society, especially among our youth. We have come to a point in this nation where we see violence everywhere. It is on the streets, in the workplace, and especially in the schools. How did this happen to our society? Is this unrivaled period of juvenile violent crime a new phenomenon? Our society demands that children have a safe environment in which to learn and grow, yet continued reports of youth violence indicates that our efforts have not been successful. Is violence prevention a myth or reality? Since children are our future, how can we provide them with the skills that will afford them the opportunity to become productive members of society?

Effects of mu- and delta-opioid-receptor antagonists on the stimulus properties of cholecystokinin.

Melton and Riley recently reported that the relatively selective mu-opioid-antagonist naloxone potentiated the stimulus properties of the gut peptide cholecystokinin (CCK). To assess whether such opioid potentiation is limited to activity at the mu-receptor subtype, in the present experiment the effects of the highly selective delta-antagonist naltrindole on CCK's stimulus properties were examined. Because in the initial report of naloxone's potentiation of CCK a relatively high, nonphysiologic dose of CCK (i.e., 13 micrograms/kg) was used as the training drug, in the current analysis subjects were trained to discriminate 5.6 micrograms/kg CCK from its vehicle and the assessments and comparisons of the effects of naloxone and naltrindole were based on this dose. Specifically, rats were administered 5.6 micrograms/kg CCK before saccharin-LiCl pairings and the CCK vehicle before saccharin alone. With such training, they rapidly acquired the drug discrimination, avoiding saccharin consumption when it was preceded by CCK and consuming the same saccharin solution when it was preceded by its vehicle. In subsequent generalization tests, doses of CCK that were ineffective in suppressing saccharin consumption (i.e., did not substitute for the training dose of CCK) did result in the suppression of saccharin consumption when combined with doses of the mu antagonist naloxone that alone had no effect on saccharin intake. On the other hand, the highly selective delta-opioid-receptor antagonist naltrindole was ineffective in potentiating the effects of CCK. Specifically, when naltrindole was combined with ineffective doses of CCK, subjects drank at control levels. The ability of naloxone to potentiate CCK's stimulus effects is consistent with a range of other demonstrations of the role of the mu-opioid-receptor subtype in CCK-opioid interactions, although the specific basis for the interaction remains unknown. Given recent findings on the effects of delta agonists and antagonists on CCK-induced activity, the failure of naltrindole to potentiate CCK's stimulus effects may be due to the absence of delta activity within this preparation, rather than the absence of delta mediation of CCK-opioid interactions in general.

Receptor mediation of the stimulus properties of cholecystokinin.

Recently, Melton, Kopman, and Riley (20) reported the rapid acquisition of drug discrimination learning using the sulfated form of cholecystokinin (CCK) within the conditioned taste aversion baseline of drug discrimination learning. The present study was designed to explore the receptor mediation of the stimulus properties of CCK within this procedure. Every fourth day, experimental subjects were given CCK-saccharin-LiCl pairings, and on the intervening recovery days, saccharin alone. Once discriminative control was established, doses of the CCK receptor antagonists devazepide (CCK-type A receptor subtype) and L-365,260 (CCK-type B receptor subtype) were administered in combination with the training dose of CCK. Unlike L-365,260 (1-1000 micrograms/kg), devazepide (1 microgram/kg) blocked the CCK stimulus, suggesting that within this design CCK's stimulus properties are mediated by the CCK-type A receptor subtype.

An assessment of the interaction between cholecystokinin and the opiates within a drug discrimination procedure.

Recently, cholecystokinin (CCK) has been reported to antagonize a variety of opiate-induced effects, including nociception, body shaking, thermoregulation, and locomotion. Consistent with these results, a number of CCK antagonists potentiate the opiates in a range of behavioral and physiological assessments. The present study further examined the interaction between CCK and the opiates within the conditioned taste aversion baseline of drug discrimination learning, a design that utilizes the stimulus properties of the drug to control consummatory behavior. Specifically, animals injected with CCK prior to saccharin-LiCl pairings and the CCK vehicle prior to saccharin alone rapidly acquired the CCK-vehicle discrimination, avoiding saccharin consumption following the administration of CCK and consuming the same saccharin solution following the vehicle. Although the stimulus properties of CCK did not generalize to either naloxone or diprenorphine, morphine blocked and naloxone potentiated CCK's stimulus effects. These data are thus consistent with a physiological (rather than a pharmacological) interaction between CCK and the opiates.

Cholecystokinin as a stimulus in drug discrimination learning.

Animals were trained to discriminate a relatively low dose of the octapeptide cholecystokinin (CCK) from distilled water within the conditioned taste aversion baseline of drug discrimination learning. Specifically, rats were injected with CCK (5.6 micrograms/kg) prior to the presentation of saccharin-LiCl pairings and with the CCK vehicle prior to the presentation of saccharin alone. After 10 conditioning trials (40 days), subjects acquired the discrimination, avoiding saccharin consumption following administration of CCK and consuming the same saccharin solution following the drug vehicle. Once the discrimination was acquired, a generalization function was determined for doses above and below that of the training stimulus. At doses below the training dose of CCK (i.e., 0, 3.2, and 4.2 micrograms/kg), subjects drank at control levels, whereas at the training dose and above (10 micrograms/kg) subjects significantly reduced consumption. That a relatively low dose of CCK can be used as a discriminative stimulus within a drug discrimination design may be important in that the procedure can now be used in the assessment of the pharmacological characteristics of CCK at a dose similar to that used in other behavioral assessments of the compound.

Gastric capacity, gastric emptying, and test-meal intake in normal and bulimic women.

The role of the stomach in regulating appetite in bulimia nervosa was examined. Subjects were nine normal and nine bulimic women of similar age, height, and weight. Gastric capacity was estimated by filling a balloon in the stomach. The mean stomach capacity of bulimic subjects was significantly larger than that of normal subjects, as revealed by the larger balloon volume tolerated (P less than 0.01) and by the larger volume needed to produce a 5 cm H2O increase in intragastric pressure (P = 0.07). The intake of a liquid meal was also significantly larger for the bulimic subjects. Gastric-emptying rate of a liquid meal was significantly delayed in the bulimic subjects during the initial 5-15 min. In all subjects, test-meal intake correlated significantly with gastric capacity (r = 0.53). In the bulimic subjects, self-reported binge intake (J) also correlated significantly with gastric capacity (r = 0.75). Binge eating in bulimic subjects may enlarge gastric capacity, which could then promote even larger binges through positive feedback.

Cholecystokinin (CCK-8) affects gastric pressure and ratings of hunger and fullness in women.

Cholecystokinin (CCK) may affect food intake by augmenting neural activity from the distended stomach, thereby amplifying satiety signals. To test the hypothesis that subjects would report more fullness and less hunger with gastric distension when CCK-8 (112 ng/min) was infused than when saline was infused, a gastric balloon was inflated in the stomachs of four women. When the balloon was inflated to 500 ml, there was no difference in gastric pressure between the CCK-8 and saline conditions. Nonetheless, ratings of fullness were higher with CCK-8 administration. When the balloon was inflated to the maximum volume tolerated, the pressure rise was significantly smaller with CCK-8 infusion. In addition, fullness ratings rose and hunger ratings declined more steeply in relation to gastric pressure when CCK-8 was infused. In all conditions, gastric contractions were practically abolished with CCK-8 infusion. CCK-8 relaxed the stomach and concurrently sensitized the subjects to gastric pressure.

A demonstration of the graded nature of the generalization function of drug discrimination learning within the conditioned taste aversion procedure.

Although control of discriminative performance will often generalize to different doses of the training drug or to drugs from the same class as the training drug, the nature of this generalization is unknown. Prior work has suggested that the generalization is primarily quantal in nature with animals displaying either vehicle-appropriate or drug-appropriate responding, depending upon their detection of the drug stimulus. It has been questioned whether this quantal nature of generalization reflects a characteristic response to drug stimuli or whether such responding is a function of the specific training and testing procedures used to establish and measure drug discrimination learning. The present paper evaluated this issue by analyzing the generalization functions of individual subjects trained and tested within one specific drug discrimination procedure, i.e. the conditioned taste aversion design. Responding within this design was generally graded. It is clear that quantal responding is not a necessary outcome of drug generalization assessments and that the nature of generalization in drug discrimination learning is a function of the specific procedure utilized in training and testing the discrimination. The results of the present analysis are discussed in terms of other recent work reporting graded functions.

Clinical trial of silicone-rubber gastric balloon to treat obesity.

A study was conducted to test the efficacy and safety of a 300 ml silicone-rubber gastric balloon for weight reduction. Eighty-six obese subjects were distributed into four groups: (1) gastric balloon only, (2) gastric balloon and prescribed 1000 kcal/day (239 kJ) diet, (3) 1000 kcal/day diet only, and (4) no treatment. The intervention period was 3 months. The balloon only group lost 3.2 kg +/- 0.9 (s.e.), the balloon and diet group lost 5.1 +/- 1.0 kg, the diet group lost 6.9 +/- 1.4 kg and the control group gained 0.6 +/- 0.5 kg. The three intervention groups each lost significantly more weight than the control group. The diet only group lost significantly more weight than the balloon only group. Body densitometry showed that the treatment groups lost a significant amount of body fat. Gastroscopy revealed three ulcers and two superficial erosions at balloon removal. The gastric balloons were well tolerated despite gastric spasms and nausea which abated after the initial 24-48 hours. Gastric capacity was determined in a subset of 19 subjects from the two balloon groups before the intervention by distending the stomach with a balloon and calculating the volume required to produce an increase in intragastric pressure of 5 cm H2O. Subjects with a smaller gastric capacity lost more weight with the balloon than subjects with a large capacity (r = 0.45, P less than 0.05). These results suggest that for improved efficacy, balloon volume may need to be larger than 300 ml or adjusted to the individual's gastric capacity.(ABSTRACT TRUNCATED AT 250 WORDS)

Gastric balloon to treat obesity: a double-blind study in nondieting subjects.

To determine its efficacy and safety in treating obesity, a silicone-rubber balloon was passed into the stomach of 10 nondieting, obese subjects. In a counterbalanced sequence, the balloon was inflated with 400 mL for 1 mo and deflated for 1 mo. Lower intakes of solid and liquid test meals (NS), significantly slower gastric emptying, and concomitant changes in glucose, insulin, glucagon, and cholecystokinin concentrations consistent with slower emptying resulted during balloon inflation. After balloon inflation, one small gastric ulcer developed, which subsequently healed. Significant weight loss occurred during the second and third week of the inflation period (F[1,9] = 5.0, p less than 0.05). However, the weight loss was small and the significant effect did not continue through the fourth week.

Evaluation of the Limulus test for endotoxemia in neonates with suspected sepsis.

A simplified test for endotoxemia was evaluated in parallel with 198 blood cultures from 115 neonates requiring transitional or intensive care. The Limulus assay disclosed endotoxemia in seven of eight patients with gram-negative bacteremia tested on one or two occasions. It was not specific for bacteremia, especially during the first week of life, when 37 of 112 tests from nonbacteremic infants (33%) were positive. In older infants, positive tests were obtained in only ten of 55 without bacteremia (18%) (P less than 0.07), six of whom had necrotizing enterocolitis as the likely source of endotoxemia. Gram-negative bacteremia existed in 5% of infants (two of 39) less than or equal to 7 days with positive tests and in 37% of older infants (six of 16) (P less than 0.01). Endotoxemia appears to be frequent among infants appearing to have sepsis and may contribute to neonatal morbidity. The Limulus assay may be a useful diagnostic test for coliform bacteremia and necrotizing enterocolitis beyond the first week of life.