DNA methylation and substance-use risk: a prospective, genome-wide study spanning gestation to adolescence.

Epigenetic processes have been implicated in addiction; yet, it remains unclear whether these represent a risk factor and/or a consequence of substance use. Here, we believe we conducted the first genome-wide, longitudinal study to investigate whether DNA methylation patterns in early life prospectively associate with substance use in adolescence. The sample comprised of 244 youth (51% female) from the Avon Longitudinal Study of Parents and Children (ALSPAC), with repeated assessments of DNA methylation (Illumina 450k array; cord blood at birth, whole blood at age 7) and substance use (tobacco, alcohol and cannabis use; age 14-18). We found that, at birth, epigenetic variation across a tightly interconnected genetic network (n=65 loci; q<0.05) associated with greater levels of substance use during adolescence, as well as an earlier age of onset amongst users. Associations were specific to the neonatal period and not observed at age 7. Key annotated genes included PACSIN1, NEUROD4 and NTRK2, implicated in neurodevelopmental processes. Several of the identified loci were associated with known methylation quantitative trait loci, and consequently likely to be under significant genetic control. Collectively, these 65 loci were also found to partially mediate the effect of prenatal maternal tobacco smoking on adolescent substance use. Together, findings lend novel insights into epigenetic correlates of substance use, highlight birth as a potentially sensitive window of biological vulnerability and provide preliminary evidence of an indirect epigenetic pathway linking prenatal tobacco exposure and adolescent substance use.

Environmental risk, Oxytocin Receptor Gene (OXTR) methylation and youth callous-unemotional traits: a 13-year longitudinal study.

Youth with high callous-unemotional traits (CU) are at risk for early-onset and persistent conduct problems. Research suggests that there may be different developmental pathways to CU (genetic/constitutional vs environmental), and that the absence or presence of co-occurring internalizing problems is a key marker. However, it is unclear whether such a distinction is valid. Intermediate phenotypes such as DNA methylation, an epigenetic modification regulating gene expression, may help to clarify etiological pathways. This is the first study to examine prospective inter-relationships between environmental risk (prenatal/postnatal) and DNA methylation (birth, age 7 and 9) in the prediction of CU (age 13), for youth low vs high in internalizing problems. We focused on DNA methylation in the vicinity of the oxytocin receptor (OXTR) gene as it has been previously implicated in CU. Participants were 84 youth with early-onset and persistent conduct problems drawn from the Avon Longitudinal Study of Parents and Children. For youth with low internalizing problems (46%), we found that (i) OXTR methylation at birth associated with higher CU (age 13) as well as decreased experience of victimization during childhood (evocative epigenetic-environment correlation; birth-age 7), (ii) higher prenatal parental risks (maternal psychopathology, criminal behaviors, substance use) associated with higher OXTR methylation at birth and (iii) OXTR methylation levels were more stable across time (birth-age 9). In contrast, for youth with high internalizing problems, CU were associated with prenatal risks of an interpersonal nature (that is, intimate partner violence, family conflict) but not OXTR methylation. Findings support the existence of distinct developmental pathways to CU.

Childhood intelligence is heritable, highly polygenic and associated with FNBP1L.

Intelligence in childhood, as measured by psychometric cognitive tests, is a strong predictor of many important life outcomes, including educational attainment, income, health and lifespan. Results from twin, family and adoption studies are consistent with general intelligence being highly heritable and genetically stable throughout the life course. No robustly associated genetic loci or variants for childhood intelligence have been reported. Here, we report the first genome-wide association study (GWAS) on childhood intelligence (age range 6-18 years) from 17,989 individuals in six discovery and three replication samples. Although no individual single-nucleotide polymorphisms (SNPs) were detected with genome-wide significance, we show that the aggregate effects of common SNPs explain 22-46% of phenotypic variation in childhood intelligence in the three largest cohorts (P=3.9 × 10(-15), 0.014 and 0.028). FNBP1L, previously reported to be the most significantly associated gene for adult intelligence, was also significantly associated with childhood intelligence (P=0.003). Polygenic prediction analyses resulted in a significant correlation between predictor and outcome in all replication cohorts. The proportion of childhood intelligence explained by the predictor reached 1.2% (P=6 × 10(-5)), 3.5% (P=10(-3)) and 0.5% (P=6 × 10(-5)) in three independent validation cohorts. Given the sample sizes, these genetic prediction results are consistent with expectations if the genetic architecture of childhood intelligence is like that of body mass index or height. Our study provides molecular support for the heritability and polygenic nature of childhood intelligence. Larger sample sizes will be required to detect individual variants with genome-wide significance.

Genome-wide association study of body mass index in 23 000 individuals with and without asthma.

BACKGROUND: Both asthma and obesity are complex disorders that are influenced by environmental and genetic factors. Shared genetic factors between asthma and obesity have been proposed to partly explain epidemiological findings of co-morbidity between these conditions. OBJECTIVE: To identify genetic variants that are associated with body mass index (BMI) in asthmatic children and adults, and to evaluate if there are differences between the genetics of BMI in asthmatics and healthy individuals. METHODS: In total, 19 studies contributed with genome-wide analysis study (GWAS) data from more than 23 000 individuals with predominantly European descent, of whom 8165 are asthmatics. RESULTS: We report associations between several DENND1B variants (P = 2.2 × 10(-7) for rs4915551) on chromosome 1q31 and BMI from a meta-analysis of GWAS data using 2691 asthmatic children (screening data). The top DENND1B single nucleotide polymorphisms(SNPs) were next evaluated in seven independent replication data sets comprising 2014 asthmatics, and rs4915551 was nominally replicated (P < 0.05) in two of the seven studies and of borderline significance in one (P = 0.059). However, strong evidence of effect heterogeneity was observed and overall, the association between rs4915551 and BMI was not significant in the total replication data set, P = 0.71. Using a random effects model, BMI was overall estimated to increase by 0.30 kg/m(2) (P = 0.01 for combined screening and replication data sets, N = 4705) per additional G allele of this DENND1BSNP. FTO was confirmed as an important gene for adult and childhood BMI regardless of asthma status. CONCLUSIONS AND CLINICAL RELEVANCE: DENND1B was recently identified as an asthma susceptibility gene in a GWAS on children, and here, we find evidence that DENND1B variants may also be associated with BMI in asthmatic children. However, the association was overall not replicated in the independent data sets and the heterogeneous effect of DENND1B points to complex associations with the studied diseases that deserve further study.

Polymorphisms in the PTPN22 region are associated with psoriasis of early onset.

BACKGROUND: Psoriasis, a chronic inflammatory skin disease, affects approximately 2% of the population worldwide. Although the aetiology of psoriasis is poorly understood, patients with disease of early onset (Type I, age of onset

Lack of association of MYO9B genetic variants with coeliac disease in a British cohort.

BACKGROUND AND AIMS: Development of coeliac disease involves an interaction between environmental factors (especially dietary wheat, rye, and barley antigens) and genetic factors (there is strong inherited disease susceptibility). The known human leucocyte antigen (HLA)-DQ2 and -DQ8 association explains only a minority of disease heritability. A recent study in the Dutch population suggested that genetic variation in the 3' region of myosin IXB (MYO9B) predisposes to coeliac disease. MYO9B is a Rho family GTPase activating protein involved in epithelial cell cytoskeletal organisation. MYO9B is hypothesised to influence intestinal permeability and hence intestinal antigen presentation. METHODS: Four single nucleotide polymorphisms were chosen to tag all common haplotypes of the MYO9B 3' haplotype block (exons 15-27). We genotyped 375 coeliac disease cases and 1366 controls (371 healthy and 995 population based). All individuals were of White UK Caucasian ethnicity. RESULTS: UK healthy control and population control allele frequencies were similar for all MYO9B variants. Case control analysis showed no significant association of any variant or haplotype with coeliac disease. CONCLUSIONS: Genetic variation in MYO9B does not have a major effect on coeliac disease susceptibility in the UK population. Differences between populations, a weaker effect size than originally described, or possibly a type I error in the Dutch study might explain these findings.

The glycocalyx of the sperm surface.

The surface of mammalian spermatozoa is covered by a dense coating of carbohydrate-rich molecules forming a 20-60 nm thick glycocalyx. The majority of sugar residues are attached to proteins which are either integrated within the sperm membrane, or are more or less loosely associated with it. It is estimated that there may be several hundred different glycoproteins comprising the glycocalyx, some of which are synthesized within the testis. Others, however, are produced by the epithelia of the efferent ducts, epididymis and possibly other accessory glands, and become associated with the spermatozoa post-testicularly during transit through, and storage in, the male tract. The acquisition of the mature glycocalyx is associated with the attainment of full sperm fertilizing ability. Until its complete molecular structure is elucidated, the complex function of the glycocalyx remains obscure, though it may be related to membrane maturation and immunoprotection in the female tract, as well as to sperm-zona binding and fertilization.

Thermal and metabolic responses to cold-water immersion at knee, hip, and shoulder levels.

To examine the effect of cold-water immersion at different depths on thermal and metabolic responses, eight men (25 yr old, 16% body fat) attempted 12 tests: immersed to the knee (K), hip (H), and shoulder (Sh) in 15 and 25 degrees C water during both rest (R) or leg cycling [35% peak oxygen uptake; (E)] for up to 135 min. At 15 degrees C, rectal (Tre) and esophageal temperatures (Tes) between R and E were not different in Sh and H groups (P > 0.05), whereas both in K group were higher during E than R (P < 0.05). At 25 degrees C, Tre was higher (P < 0.05) during E than R at all depths, whereas Tes during E was higher than during R in H and K groups. Tre remained at control levels in K-E at 15 degrees C, K-E at 25 degrees C, and in H-E groups at 25 degrees C, whereas Tes remained unchanged in K-E at 15 degrees C, in K-R at 15 degrees C, and in all 25 degrees C conditions (P > 0.05). During R and E, the magnitude of Tre change was greater (P < 0.05) than the magnitude of Tes change in Sh and H groups, whereas it was not different in the K group (P > 0.05). Total heat flow was progressive with water depth. During R at 15 and 25 degrees C, heat production was not increased in K and H groups from control level (P > 0.05) but it did increase in Sh group (P < 0.05). The increase in heat production during E compared with R was smaller (P < 0.05) in Sh (121 +/- 7 W/m2 at 15 degrees C and 97 +/- 6 W/m2 at 25 degrees C) than in H (156 +/- 6 and 126 +/- 5 W/m2, respectively) and K groups (155 +/- 4 and 165 +/- 6 W/m2, respectively). These data suggest that Tre and Tes respond differently during partial cold-water immersion. In addition, water levels above knee in 15 degrees C and above hip in 25 degrees C cause depression of internal temperatures mainly due to insufficient heat production offsetting heat loss even during light exercise.

Thermal responses of men and women during cold-water immersion: influence of exercise intensity.

The influence of exercise intensity on thermoregulation was studied in 8 men and 8 women volunteers during three levels of arm-leg exercise (level I: 700 ml oxygen (O2).min-1; level II: 1250 ml O2.min-1; level III: 1700 ml O2.min-1) for 1 h in water at 20 and 28 degrees C (Tw). For the men in Tw 28 degrees C the rectal temperature (Tre) fell 0.79 degree C (P less than 0.05) during immersion in both rest and level-I exercise. With level-II exercise a drop in Tre of 0.54 degree C (P less than 0.05) was noted, while at level-III exercise Tre did not change from the pre-immersion value. At Tw of 20 degrees C, Tre fell throughout immersion with no significant difference in final Tre observed between rest and any exercise level. For the women at rest at Tw 28 degrees C, Tre fell 0.80 degree C (P less than 0.05) below the pre-immersion value. With the two more intense levels of exercise Tre did not decrease during immersion. In Tw 20 degrees C, the women maintained higher Tre (P less than 0.05) during level-II and level-III exercise compared to rest and exercise at level I. The Tre responses were related to changes in tissue insulation (I(t)) between rest and exercise with the largest reductions in I(t) noted between rest and level-I exercise across Tw and gender. For mean and women of similar percentage body fat, decreases in Tre were greater for the women at rest and level-I exercise in Tw 20 degrees C (P less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

Lipid and lipoprotein profiles relate to peak aerobic power in spinal cord injured men.

The relationship between peak VO2 and serum lipids, lipoproteins, and apolipoproteins was assessed in nine traumatic spinal cord injured (SCI), active, male volunteers. Mean (SD) age, height, and weight were 30.6 (11.6) yr, 171.1 (11.3) cm, and 74.2 (12.5) kg, respectively. Peak VO2 (X = 2.13 l.min-1) was assessed by a graded arm-crank test to maximum, percent body fat (X = 28.7%) by densitometry, and lipid profile by measures of serum triglycerides, total cholesterol (TC), high and low density lipoprotein cholesterol (HDL-C and LDL-C, respectively), apolipoproteins (apoA-1, apoB), and various ratios of lipids, lipoproteins, and apolipoproteins. Significant inverse relationships emerged between peak VO2 and TC/HDL-C (r = -0.86; P less than 0.01), apoB/apoA-1 (r = -0.75; P less than 0.05), triglycerides (r = -0.73; P less than 0.05), and LDL-C/HDL-C (r = -0.72; P less than 0.05). Direct correlations (P less than 0.05) were demonstrated between peak VO2 and apoA-1/apoB (r = 0.71) and HDL-C/apoA-1 (r = 0.64). The present results indicate that, for active, mid-to-lower thoracic SCI men, the putative atherogenic and antiatherogenic lipid, lipoprotein, and apolipoprotein indices are significantly related to peak VO2 in a manner similar to that described for the able-bodied. These findings indicate the relevance of aerobic fitness assessment in planning CHD prevention strategies for the SCI.

Cardiovascular adjustments to exercise distributed between the upper and lower body.

The present study examined the hemodynamic differences between upper- and lower-body exercise where the total power output (PO) was proportionally distributed between the upper and lower body. Six males completed five combinations of arm-leg exercise at maximal and three submaximal intensities. The ratio of arm PO to total PO for each exercise combination was 0, 25, 50, 75, and 100%. At each submaximal intensity, VO2 and cardiac output (Q) were not different (P greater than 0.05) across exercise combinations. Likewise, heart rate (HR) responses were not different for 0, 25, 50, and 75% at level 1 (mean = 102, 102, 106, 106 beats.min-1, respectively), level 2 (mean = 114, 110, 119, 118 beats.min-1, respectively), and level 3 (mean = 127, 124, 132, 131 beats.min-1, respectively). However, HR for 100% (arm-only exercise) tended to be higher than 0% at level 1 (delta HR = 10 beats.min-1; P less than 0.10), level 2 (delta HR = 12 beats.min-1, P less than 0.06) and level 3 (delta HR = 10 beats.min-1; P less than 0.06). At level 1, stroke volume (SV) remained essentially unchanged from 0-75%, while SV at 100% (108 ml) was slightly though not significantly lower (P less than 0.10) than 0% (125 ml). At exercise levels 2 and 3, SV remained unchanged for 0 and 25%; however, SV at 50, 75, and 100% were generally lower (P less than 0.05) compared with 0%. These results indicate that involving the leg musculature to varying degrees during arm-leg exercise attenuates the hemodynamic differences observed during strict upper body versus strict lower body exercise.

Beta-adrenergic-receptor-mediated suppression of interleukin 2 receptors in human lymphocytes.

Adrenergic receptor agonists are known to attenuate the proliferative response of human lymphocytes after activation; however, their mechanism of action is unknown. Since expression of interleukin 2 (IL-2) receptors is a prerequisite for proliferation, the effect of beta-adrenergic receptor agonists on lymphocyte IL-2 receptors was studied on both mitogen-stimulated lymphocytes and IL-2-dependent T lymphocyte cell lines. In both cell types the beta-adrenergic receptor agonist isoproterenol blocked the expression of IL-2 receptors, as determined with the IL-2 receptor anti-TAC antibody. To determine the effect of beta-adrenergic agonists on expression of the high affinity IL-2 receptors, [125I]IL-2 binding studies were performed at concentrations selective for high affinity sites. No significant effect of beta-adrenergic agonists on high affinity IL-2 receptor sites could be detected. The data demonstrate that beta-adrenergic receptor agonists down-regulate IL-2 receptors primarily affecting low affinity sites.

Are lymphocyte beta-adrenoceptors altered in patients with cystic fibrosis?

1. Beta-adrenergic responsiveness may be decreased in cystic fibrosis. In order to determine whether this reflects an alteration in the human lymphocyte beta-receptor complex, we studied 12 subjects with cystic fibrosis (six were stable and ambulatory and six were decompensated, hospitalized) as compared with 12 normal controls. 2. Lymphocyte beta-receptor mediated adenylate cyclase activity (EC 4.6.1.1) was not decreased in the ambulatory cystic fibrosis patients as compared with controls. In contrast, decompensated hospitalized cystic fibrosis patients demonstrated a significant reduction in beta-receptor mediated lymphocyte adenylate cyclase activity expressed as the relative increase over basal levels stimulated by the beta-agonist isoprenaline compared with both normal controls and stable ambulatory cystic fibrosis patients (control 58 +/- 4%; ambulatory cystic fibrosis patients 51 +/- 7%; decompensated hospitalized cystic fibrosis patients 28 +/- 5%; P less than 0.05). 3. Our data suggest that defects in lymphocyte beta-receptor properties in cystic fibrosis patients may be better correlated with clinical status than with presence or absence of the disease state.

Low sodium diet corrects the defect in lymphocyte beta-adrenergic responsiveness in hypertensive subjects.

To determine the role of dietary sodium intake in the reduction in beta-adrenergic sensitivity in hypertension, lymphocyte beta-receptors from 8 borderline hypertensive and 16 normotensive subjects were studied after 5 d on a high sodium diet (400 meq/d) and also following a low sodium diet (10 meq/d). During the high sodium diet, lymphocyte beta-receptor-stimulated adenylate cyclase activity, expressed as the relative increase over basal levels stimulated by the beta-agonist isoproterenol, was significantly (P less than 0.025) decreased in hypertensive (24 +/- 5%, mean +/- SE) compared with normotensive (42 +/- 4%) subjects. Neither beta-receptor density nor the proportion of nonsequestered beta-receptors differed between groups. A low sodium diet significantly increased beta-receptor-stimulated adenylate cyclase activity in hypertensives (low sodium, 51 +/- 7%; high sodium, 24 +/- 5%, P less than 0.025) to a level not different than that of normotensives (46 +/- 5%). Thus, reduced lymphocyte beta-receptor responsiveness in hypertensive subjects is not due to beta-receptor sequestration and is corrected on a low sodium diet. Dietary sodium may be an important factor in the beta-receptor defect in early hypertension.

Phenylarsine oxide inhibits agonist-induced changes in photolabeling but not agonist-induced desensitization of the beta-adrenergic receptor.

In the human lymphocyte, desensitization of the beta-adrenergic receptor-adenylate cyclase complex is associated with sequestration of the receptor as well as a change in photolabeling of beta-receptor proteins. Thus, desensitization of the lymphocyte beta-adrenergic receptor-adenylate cyclase system is associated with a selective reduction in the photoaffinity labeling of an Mr approximately equal to 55,000 beta-adrenergic receptor-binding site as compared to an Mr approximately equal to 68,000 beta-adrenergic receptor-binding moiety. In order to examine the relationship between sequestration and reduction in labeling of the Mr approximately equal to 55,000 peptide, we have studied the effect of phenylarsine oxide (an inhibitor of beta-receptor sequestration in astrocytoma cells) on agonist-induced desensitization of the beta-adrenergic receptor-adenylate cyclase system in circulating lymphocytes. Incubation of cells with phenylarsine oxide prior to exposure to agonists did not block the consequent reduction in isoproterenol-stimulated adenylate cyclase activity. However, sequestration of the receptor, as assessed by a decrease in accessibility of beta-adrenergic receptors on intact cells to hydrophilic receptor ligands, is blocked by phenylarsine oxide. Thus, the agonist-induced reduction in binding of the hydrophilic beta-adrenergic receptor ligand CGP-12177 was blocked by phenylarsine oxide (without phenylarsine oxide, 57 +/- 6% of control, with phenylarsine oxide, 97 +/- 3% of control). Photolabeling studies with [125I]iodocyanopindolol diazirine revealed that phenylarsine oxide pretreatment also blocked the selective loss in labeling of the Mr approximately equal to 55,000 beta-adrenergic receptor protein. These data suggest that agonist-induced alterations in the photolabeling pattern of the lymphocyte beta-adrenergic receptor that occur with desensitization closely parallel the apparent sequestration of beta-adrenergic receptors but can be dissociated from the initial desensitization phenomenon.

Release of catecholamines from superfused bovine adrenal chromaffin cells cultured on microcarrier beads.

A procedure is described for the establishment of stable primary cultures of bovine chromaffin cells on microcarrier beads. The cells flatten and send out processes with varicosities over a few days and maintain their catecholamine content for 2 weeks. The beads may be incorporated into a superfusion apparatus with a chamber volume of about 150 microliters, enabling the efficient perfusion of a high density of cells. The response to the introduction of nicotine and high potassium into the perfusing medium is shown to be more rapid and more transient than hitherto described, with each secretagogue producing a different degree of preferential stimulation of noradrenaline-secreting cells.

Breakdown of small enkephalin derivatives and adrenal peptide E by human plasma.

To provide information concerning the fate of opioid peptides introduced into the circulation of man, we have investigated the breakdown of the following peptides when incubated in human plasma: [Met]enkephalin, [Met]enkephalyl-Arg6-Phe7, [Met]enkephalyl-Arg6-Gly7-Leu8, [Met]enkephalyl-Arg6-Arg7-Val8-NH2 and the complex opioid, peptide E. We used a radioimmunoassay recognizing the amino-terminus of opioid peptides (assay for total opioid peptide-like immunoreactivity). The three small enkephalin derivatives were broken down considerably faster than the enkephalins themselves. The rate of loss of immunoreactivity was considerably reduced by bacitracin. When peptide E was incubated in human plasma, a relatively sustained level of opioid peptide-like immunoreactivity was seen. This was shown to be due, not only to the relatively slow aminopeptidase attack of the larger peptide, but also to the generation during breakdown of peptide E of [Leu]enkephalin and [Met]-enkephalin.

Opioid peptides in human adrenal: partial characterization and presence of adrenal peptide E.

Evaluation of hypotheses concerning the role of opioid peptides in the human adrenal is handicapped by the lack of information concerning the nature of these peptides. We studied the content of opioid peptides in whole adrenal tissue using several RIAs, including one which cross-reacts with all opioid peptides tested. Opioid peptides were localized to granules which behaved like chromaffin granules on crude sucrose density separation. beta-Endorphin immunoreactivity was a minor component, which was found principally in the form of beta-endorphin-(1-31). The majority of the remaining peptides probably were products of proenkephalin. The human postmortem tissue differed from bovine tissue in that the major accumulating products of this precursor were the size of the enkephalins and their small congeners, and not the intermediate-sized (mol wt, approximately 1500-4000) peptides that predominated in bovine tissue. We also found evidence of the presence of the 25-amino acid complex opioid, peptide E, in human tissue.

Metorphamide, a C-terminally amidated opioid peptide, in human adrenal and human phaeochromocytoma.

There appears to be only one possible site for the production of an amidated peptide in the human proenkephalin sequence; this will give rise to the peptide named metorphamide. Since amidation of peptides is commonly an activation step in the synthesis of regulatory peptides, we have examined the levels and form of immunoreactivity to metorphamide in human post-mortem adrenal and phaeochromocytoma extracts. In three out of four post-mortem adrenal extracts, and in each of the two phaeochromocytoma extracts examined, there was 3-4 times more immunoreactivity to the carboxy-terminus of pro-enkephalin, Met-enkephalin(Arg6,Phe7), than to metorphamide. The metorphamide immunoreactivity was shown in each extract to measure only the amidated octapeptide according to gel exclusion and reverse-phase chromatography data. The implications for processing of proenkephalin in human adrenal are indicated.