Postprandial alterations in whole-blood DNA methylation are mediated by changes in white blood cell composition.

BACKGROUND: DNA methylation is an essential nuclear process associated with genomic functions such as transcription factor binding and the regulation of gene expression. DNA methylation patterns can also serve as potential biomarkers for disease progression and response to therapy. However, the full dynamics of DNA methylation across daily physiologic events have not been fully elucidated. OBJECTIVE: We sought to study how ingesting a standardized meal acutely affects peripheral blood DNA methylation. DESIGN: We performed an observational study in healthy men (n = 26) on DNA methylation and gene expression in whole blood before and 160 min after the ingestion of a standardized meal. Cytosine-phosphate-guanine (CpG) methylation was assayed on the HumanMethylation450k microarray, and gene expression was measured with the Human Gene 2.1 ST Array. RESULTS: Differential methylation after food intake was detected in 13% of the analyzed probes (63,207 CpG probes) at a 5% false discovery rate (FDR). This effect was driven by changes in leukocyte fractions as estimated from comparisons against methylation datasets generated from sorted leukocytes. When methylation values were adjusted for estimated leukocyte fractions, 541 probes were observed to be altered in the postprandial state (5% FDR). CONCLUSIONS: Apparent alterations in DNA methylation 160 min after meal ingestion mainly reflect changes in the estimated leukocyte population in whole blood. These results have major methodologic implications for genome-wide methylation studies because they highlight the strong underlying effects of changes in leukocyte fractions on CpG methylation patterns as well as the potential importance of meal-standardized sampling procedures for future investigations when alterations in white blood cell fractions are unavailable. This trial was registered at clinicaltrials.gov as LSF008786.

Epigenomics of Total Acute Sleep Deprivation in Relation to Genome-Wide DNA Methylation Profiles and RNA Expression.

Despite an established link between sleep deprivation and epigenetic processes in humans, it remains unclear to what extent sleep deprivation modulates DNA methylation. We performed a within-subject randomized blinded study with 16 healthy subjects to examine the effect of one night of total sleep deprivation (TSD) on the genome-wide methylation profile in blood compared with that in normal sleep. Genome-wide differences in methylation between both conditions were assessed by applying a paired regression model that corrected for monocyte subpopulations. In addition, the correlations between the methylation of genes detected to be modulated by TSD and gene expression were examined in a separate, publicly available cohort of 10 healthy male donors (E-GEOD-49065). Sleep deprivation significantly affected the DNA methylation profile both independently and in dependency of shifts in monocyte composition. Our study detected differential methylation of 269 probes. Notably, one CpG site was located 69 bp upstream of ING5, which has been shown to be differentially expressed after sleep deprivation. Gene set enrichment analysis detected the Notch and Wnt signaling pathways to be enriched among the differentially methylated genes. These results provide evidence that total acute sleep deprivation alters the methylation profile in healthy human subjects. This is, to our knowledge, the first study that systematically investigated the impact of total acute sleep deprivation on genome-wide DNA methylation profiles in blood and related the epigenomic findings to the expression data.

Resting-State Brain and the FTO Obesity Risk Allele: Default Mode, Sensorimotor, and Salience Network Connectivity Underlying Different Somatosensory Integration and Reward Processing between Genotypes.

Single-nucleotide polymorphisms (SNPs) of the fat mass and obesity associated (FTO) gene are linked to obesity, but how these SNPs influence resting-state neural activation is unknown. Few brain-imaging studies have investigated the influence of obesity-related SNPs on neural activity, and no study has investigated resting-state connectivity patterns. We tested connectivity within three, main resting-state networks: default mode (DMN), sensorimotor (SMN), and salience network (SN) in 30 male participants, grouped based on genotype for the rs9939609 FTO SNP, as well as punishment and reward sensitivity measured by the Behavioral Inhibition (BIS) and Behavioral Activation System (BAS) questionnaires. Because obesity is associated with anomalies in both systems, we calculated a BIS/BAS ratio (BBr) accounting for features of both scores. A prominence of BIS over BAS (higher BBr) resulted in increased connectivity in frontal and paralimbic regions. These alterations were more evident in the obesity-associated AA genotype, where a high BBr was also associated with increased SN connectivity in dopaminergic circuitries, and in a subnetwork involved in somatosensory integration regarding food. Participants with AA genotype and high BBr, compared to corresponding participants in the TT genotype, also showed greater DMN connectivity in regions involved in the processing of food cues, and in the SMN for regions involved in visceral perception and reward-based learning. These findings suggest that neural connectivity patterns influence the sensitivity toward punishment and reward more closely in the AA carriers, predisposing them to developing obesity. Our work explains a complex interaction between genetics, neural patterns, and behavioral measures in determining the risk for obesity and may help develop individually-tailored strategies for obesity prevention.

A Genetic Risk Score Is Associated with Weight Loss Following Roux-en Y Gastric Bypass Surgery.

BACKGROUND: Currently, Roux-en Y gastric bypass (RYGB) is the most efficient therapy for severe obesity. Weight loss after surgery is, however, highly variable and genetically influenced. Genome-wide association studies have identified several single nucleotide polymorphisms (SNP) associated with body mass index (BMI) and waist-hip ratio (WHR). We aimed to identify two genetic risk scores (GRS) composed of weighted BMI and WHR-associated SNPs to estimate their impact on excess BMI loss (EBMIL) after RYGB surgery. METHODS: Two hundred and thirty-eight obese patients (BMI 45.1 ± 6.2 kg/m(2), 74 % women), who underwent RYGB, were genotyped for 35 BMI and WHR-associated SNPs and were followed up after 2 years. SNPs with high impact on post-surgical weight loss were filtered out using a random forest model. The filtered SNPs were combined into a GRS and analyzed in a linear regression model. RESULTS: An up to 11 % lower EBMIL with higher risk score was estimated for two GRS models (P = 0.026 resp. P = 0.021) composed of seven BMI-associated SNPs (closest genes: MC4R, TMEM160, PTBP2, NUDT3, TFAP2B, ZNF608, MAP2K5, GNPDA2, and MTCH2) and of three WHR-associated SNPs (closest genes: HOXC13, LYPLAL1, and DNM3-PIGC). Patients within the lowest GRS quartile had higher EBMIL compared to patients within the other three quartiles in both models. CONCLUSIONS: We identified two GRSs composed of BMI and WHR-associated SNPs with significant impact on weight loss after RYGB surgery using random forest analysis as a SNP selection tool. The GRS may be useful to pre-surgically evaluate the risks for patients undergoing RYGB surgery.

An obesity-associated risk allele within the FTO gene affects human brain activity for areas important for emotion, impulse control and reward in response to food images.

Understanding how genetics influences obesity, brain activity and eating behaviour will add important insight for developing strategies for weight-loss treatment, as obesity may stem from different causes and as individual feeding behaviour may depend on genetic differences. To this end, we examined how an obesity risk allele for the FTO gene affects brain activity in response to food images of different caloric content via functional magnetic resonance imaging (fMRI). Thirty participants homozygous for the rs9939609 single nucleotide polymorphism were shown images of low- or high-calorie food while brain activity was measured via fMRI. In a whole-brain analysis, we found that people with the FTO risk allele genotype (AA) had increased activity compared with the non-risk (TT) genotype in the posterior cingulate, cuneus, precuneus and putamen. Moreover, higher body mass index in the AA genotype was associated with reduced activity to food images in areas important for emotion (cingulate cortex), but also in areas important for impulse control (frontal gyri and lentiform nucleus). Lastly, we corroborate our findings with behavioural scales for the behavioural inhibition and activation systems. Our results suggest that the two genotypes are associated with differential neural processing of food images, which may influence weight status through diminished impulse control and reward processing.

Many obesity-associated SNPs strongly associate with DNA methylation changes at proximal promoters and enhancers.

BACKGROUND: The mechanisms by which genetic variants, such as single nucleotide polymorphisms (SNPs), identified in genome-wide association studies act to influence body mass remain unknown for most of these SNPs, which continue to puzzle the scientific community. Recent evidence points to the epigenetic and chromatin states of the genome as having important roles. METHODS: We genotyped 355 healthy young individuals for 52 known obesity-associated SNPs and obtained DNA methylation levels in their blood using the Illumina 450 K BeadChip. Associations between alleles and methylation at proximal cytosine residues were tested using a linear model adjusted for age, sex, weight category, and a proxy for blood cell type counts. For replication in other tissues, we used two open-access datasets (skin fibroblasts, n = 62; four brain regions, n = 121-133) and an additional dataset in subcutaneous and visceral fat (n = 149). RESULTS: We found that alleles at 28 of these obesity-associated SNPs associate with methylation levels at 107 proximal CpG sites. Out of 107 CpG sites, 38 are located in gene promoters, including genes strongly implicated in obesity (MIR148A, BDNF, PTPMT1, NR1H3, MGAT1, SCGB3A1, HOXC12, PMAIP1, PSIP1, RPS10-NUDT3, RPS10, SKOR1, MAP2K5, SIX5, AGRN, IMMP1L, ELP4, ITIH4, SEMA3G, POMC, ADCY3, SSPN, LGR4, TUFM, MIR4721, SULT1A1, SULT1A2, APOBR, CLN3, SPNS1, SH2B1, ATXN2L, and IL27). Interestingly, the associated SNPs are in known eQTLs for some of these genes. We also found that the 107 CpGs are enriched in enhancers in peripheral blood mononuclear cells. Finally, our results indicate that some of these associations are not blood-specific as we successfully replicated four associations in skin fibroblasts. CONCLUSIONS: Our results strongly suggest that many obesity-associated SNPs are associated with proximal gene regulation, which was reflected by association of obesity risk allele genotypes with differential DNA methylation. This study highlights the importance of DNA methylation and other chromatin marks as a way to understand the molecular basis of genetic variants associated with human diseases and traits.

Methylation levels of SLC23A2 and NCOR2 genes correlate with spinal muscular atrophy severity.

Spinal muscular atrophy (SMA) is a monogenic neurodegenerative disorder subdivided into four different types. Whole genome methylation analysis revealed 40 CpG sites associated with genes that are significantly differentially methylated between SMA patients and healthy individuals of the same age. To investigate the contribution of methylation changes to SMA severity, we compared the methylation level of found CpG sites, designed as "targets", as well as the nearest CpG sites in regulatory regions of ARHGAP22, CDK2AP1, CHML, NCOR2, SLC23A2 and RPL9 in three groups of SMA patients. Of notable interest, compared to type I SMA male patients, the methylation level of a target CpG site and one nearby CpG site belonging to the 5'UTR of SLC23A2 were significantly hypomethylated 19-22% in type III-IV patients. In contrast to type I SMA male patients, type III-IV patients demonstrated a 16% decrease in the methylation levels of a target CpG site, belonging to the 5'UTR of NCOR2. To conclude, this study validates the data of our previous study and confirms significant methylation changes in the SLC23A2 and NCOR2 regulatory regions correlates with SMA severity.

Roux-en Y gastric bypass surgery induces genome-wide promoter-specific changes in DNA methylation in whole blood of obese patients.

CONTEXT: DNA methylation has been proposed to play a critical role in many cellular and biological processes. OBJECTIVE: To examine the influence of Roux-en-Y gastric bypass (RYGB) surgery on genome-wide promoter-specific DNA methylation in obese patients. Promoters are involved in the initiation and regulation of gene transcription. METHODS: Promoter-specific DNA methylation in whole blood was measured in 11 obese patients (presurgery BMI >35 kg/m(2), 4 females), both before and 6 months after RYGB surgery, as well as once only in a control group of 16 normal-weight men. In addition, body weight and fasting plasma glucose were measured after an overnight fast. RESULTS: The mean genome-wide distance between promoter-specific DNA methylation of obese patients at six months after RYGB surgery and controls was shorter, as compared to that at baseline (p<0.001). Moreover, postsurgically, the DNA methylation of 51 promoters was significantly different from corresponding values that had been measured at baseline (28 upregulated and 23 downregulated, P<0.05 for all promoters, Bonferroni corrected). Among these promoters, an enrichment for genes involved in metabolic processes was found (n = 36, P<0.05). In addition, the mean DNA methylation of these 51 promoters was more similar after surgery to that of controls, than it had been at baseline (P<0.0001). When controlling for the RYGB surgery-induced drop in weight (-24% of respective baseline value) and fasting plasma glucose concentration (-16% of respective baseline value), the DNA methylation of only one out of 51 promoters (~2%) remained significantly different between the pre-and postsurgery time points. CONCLUSIONS: Epigenetic modifications are proposed to play an important role in the development of and predisposition to metabolic diseases, including type II diabetes and obesity. Thus, our findings may form the basis for further investigations to unravel the molecular effects of gastric bypass surgery. CLINICAL TRIAL: ClinicalTrials.gov NCT01730742.

Genome-wide analysis reveals DNA methylation markers that vary with both age and obesity.

The combination of the obesity epidemic and an aging population presents growing challenges for the healthcare system. Obesity and aging are major risk factors for a diverse number of diseases and it is of importance to understand their interaction and the underlying molecular mechanisms. Herein the authors examined the methylation levels of 27578 CpG sites in 46 samples from adult peripheral blood. The effect of obesity and aging was ascertained with general linear models. More than one hundred probes were correlated to aging, nine of which belonged to the KEGG group map04080. Additionally, 10 CpG sites had diverse methylation profiles in obese and lean individuals, one of which was the telomerase catalytic subunit (TERT). In eight of ten cases the methylation change was reverted between obese and lean individuals. One region proved to be differentially methylated with obesity (LINC00304) independent of age. This study provides evidence that obesity influences age driven epigenetic changes, which provides a molecular link between aging and obesity. This link and the identified markers may prove to be valuable biomarkers for the understanding of the molecular basis of aging, obesity and associated diseases.

Acute sleep deprivation in healthy young men: impact on population diversity and function of circulating neutrophils.

Lack of sleep greatly affects our immune system. The present study investigates the acute effects of total sleep deprivation on blood neutrophils, the most abundant immune cell in our circulation and the first cell type recruited to sites of infection. Thus, the population diversity and function of circulating neutrophils were compared in healthy young men following one night of total sleep deprivation (TSD) or after 8h regular sleep. We found that neutrophil counts were elevated after nocturnal wakefulness (2.0 ± 0.2 × 10(9)/l vs. 2.6 ± 0.2 × 10(9)/l, sleep vs. TSD, respectively) and the population contained more immature CD16(dim)/CD62L(bright) cells (0.11 ± 0.040 × 10(9)/l [5.5 ± 1.1%] vs. 0.26 ± 0.020 × 10(9)/l [9.9 ± 1.4%]). As the rise in numbers of circulating mature CD16(bright)/CD62L(bright) neutrophils was less pronounced, the fraction of this subpopulation showed a significant decrease (1.8 ± 0.15 × 10(9)/l [88 ± 1.8%] vs. 2.1 ± 0.12 × 10(9)/l [82 ± 2.8%]). The surface expression of receptors regulating mobilization of neutrophils from bone marrow was decreased (CXCR4 and CD49d on immature neutrophils; CXCR2 on mature neutrophils). The receptor CXCR2 is also involved in the production of reactive oxygen species (ROS), and in line with this, total neutrophils produced less ROS. In addition, following sleep loss, circulating neutrophils exhibited enhanced surface levels of CD11b, which indicates enhanced granular fusion and concomitant protein translocation to the membrane. Our findings demonstrate that sleep loss exerts significant effects on population diversity and function of circulating neutrophils in healthy men. To which extent these changes could explain as to why people with poor sleep patterns are more susceptible to infections warrants further investigation.

Acute sleep deprivation increases serum levels of neuron-specific enolase (NSE) and S100 calcium binding protein B (S-100B) in healthy young men.

STUDY OBJECTIVES: To investigate whether total sleep deprivation (TSD) affects circulating concentrations of neuron-specific enolase (NSE) and S100 calcium binding protein B (S-100B) in humans. These factors are usually found in the cytoplasm of neurons and glia cells. Increasing concentrations of these factors in blood may be therefore indicative for either neuronal damage, impaired blood brain barrier function, or both. In addition, amyloid ß (Aß) peptides 1-42 and 1-40 were measured in plasma to calculate their ratio. A reduced plasma ratio of Aß peptides 1-42 to 1-40 is considered an indirect measure of increased deposition of Aß 1-42 peptide in the brain. DESIGN: Subjects participated in two conditions (including either 8-h of nocturnal sleep [22:30-06:30] or TSD). Fasting blood samples were drawn before and after sleep interventions (19:30 and 07:30, respectively). SETTING: Sleep laboratory. PARTICIPANTS: 15 healthy young men. RESULTS: TSD increased morning serum levels of NSE (P = 0.002) and S-100B (P = 0.02) by approximately 20%, compared with values obtained after a night of sleep. In contrast, the ratio of Aß peptides 1-42 to 1-40 did not differ between the sleep interventions. CONCLUSIONS: Future studies in which both serum and cerebrospinal fluid are sampled after sleep loss should elucidate whether the increase in serum neuron-specific enolase and S100 calcium binding protein B is primarily caused by neuronal damage, impaired blood brain barrier function, or is just a consequence of increased gene expression in non-neuronal cells, such as leukocytes.

BDNF polymorphisms are linked to poorer working memory performance, reduced cerebellar and hippocampal volumes and differences in prefrontal cortex in a Swedish elderly population.

BACKGROUND: Brain-derived neurotrophic factor (BDNF) links learning, memory and cognitive decline in elderly, but evidence linking BDNF allele variation, cognition and brain structural differences is lacking. METHODS: 367 elderly Swedish men (n = 181) and women (n = 186) from Prospective Investigation of the Vasculature in Uppsala seniors (PIVUS) were genotyped and the BDNF functional rs6265 SNP was further examined in subjects who completed the Trail Making Task (TMT), verbal fluency task, and had a magnetic resonance imaging (MRI) scan. Voxel-based morphometry (VBM) examined brain structure, cognition and links with BDNF. RESULTS: The functional BDNF SNP (rs6265,) predicted better working memory performance on the TMT with positive association of the Met rs6265, and was linked with greater cerebellar, precuneus, left superior frontal gyrus and bilateral hippocampal volume, and reduced brainstem and bilateral posterior cingulate volumes. CONCLUSIONS: The functional BDNF polymorphism influences brain volume in regions associated with memory and regulation of sensorimotor control, with the Met rs6265 allele potentially being more beneficial to these functions in the elderly.

Acute sleep deprivation increases food purchasing in men.

OBJECTIVE: To investigate if acute sleep deprivation affects food purchasing choices in a mock supermarket. DESIGN AND METHODS: On the morning after one night of total sleep deprivation (TSD) or after one night of sleep, 14 normal-weight men were given a fixed budget (300 SEK-approximately 50 USD). They were instructed to purchase as much as they could out of a possible 40 items, including 20 high-caloric foods (>2 kcal/g) and 20 low-caloric foods (<2 kcal/g). The prices of the high-caloric foods were then varied (75%, 100% (reference price), and 125%) to determine if TSD affects the flexibility of food purchasing. Before the task, participants received a standardized breakfast, thereby minimizing the potential confound produced by hunger. In addition, morning plasma concentrations of the orexigenic hormone ghrelin were measured under fasting conditions. RESULTS: Independent of both type of food offered and price condition, sleep-deprived men purchased significantly more calories (+9%) and grams (+18%) of food than they did after one night of sleep (both P < 0.05). Morning plasma ghrelin concentrations were also higher after TSD (P < 0.05). However, this increase did not correlate with the effects of TSD on food purchasing. CONCLUSIONS: This experiment demonstrates that acute sleep loss alters food purchasing behavior in men.