Altered pathways in methylome and transcriptome longitudinal analysis of normal weight and bariatric surgery women.

DNA methylation could provide a link between environmental, genetic factors and weight control and can modify gene expression pattern. This study aimed to identify genes, which are differentially expressed and methylated depending on adiposity state by evaluating normal weight women and obese women before and after bariatric surgery (BS). We enrolled 24 normal weight (BMI: 22.5 ± 1.6 kg/m2) and 24 obese women (BMI: 43.3 ± 5.7 kg/m2) submitted to BS. Genome-wide methylation analysis was conducted using Infinium Human Methylation 450 BeadChip (threshold for significant CpG sites based on delta methylation level with a minimum value of 5%, a false discovery rate correction (FDR) of q < 0.05 was applied). Expression levels were measured using HumanHT-12v4 Expression BeadChip (cutoff of p ≤ 0.05 and fold change ≥2.0 was used to detect differentially expressed probes). The integrative analysis of both array data identified four genes (i.e. TPP2, PSMG6, ARL6IP1 and FAM49B) with higher methylation and lower expression level in pre-surgery women compared to normal weight women: and two genes (i.e. ZFP36L1 and USP32) that were differentially methylated after BS. These methylation changes were in promoter region and gene body. All genes are related to MAPK cascade, NIK/NF-kappaB signaling, cellular response to insulin stimulus, proteolysis and others. Integrating analysis of DNA methylation and gene expression evidenced that there is a set of genes relevant to obesity that changed after BS. A gene ontology analysis showed that these genes were enriched in biological functions related to adipogenesis, orexigenic, oxidative stress and insulin metabolism pathways. Also, our results suggest that although methylation plays a role in gene silencing, the majority of effects were not correlated.

DNA methylation screening after roux-en Y gastric bypass reveals the epigenetic signature stems from genes related to the surgery per se.

BACKGROUND/OBJECTIVES: Obesity has been associated with gene methylation regulation. Recent studies have shown that epigenetic signature plays a role in metabolic homeostasis after Roux-en Y gastric bypass (RYGB). To conduct a genome-wide epigenetic analysis in peripheral blood to investigate whether epigenetic changes following RYGB stem from weight loss or the surgical procedure per se. SUBJECTS/METHODS: By means of the Infinium Human Methylation 450 BeadChip array, global methylation was analyzed in blood of 24 severely obese women before and 6 months after RYGB and in 24 normal-weight women (controls). RESULTS: In blood cells, nine DMCpG sites showed low methylation levels before surgery, methylation levels increased after RYGB and neared the levels measured in the controls. Additionally, 44 CpG sites associated with the Wnt and p53 signaling pathways were always differently methylated in the severely obese patients as compared to the controls and were not influenced by RYGB. Finally, 1638 CpG sites related to inflammation, angiogenesis, and apoptosis presented distinct methylation in the post-surgery patients as compared to the controls. CONCLUSION: Bariatric surgery per se acts on CpGs related to inflammation, angiogenesis, and endothelin-signaling. However, the gene cluster associated with obesity remains unchanged, suggesting that weight loss 6 months after RYGB surgery cannot promote this effect.

DNA methylation map in circulating leukocytes mirrors subcutaneous adipose tissue methylation pattern: a genome-wide analysis from non-obese and obese patients.

The characterization of the epigenetic changes within the obesity-related adipose tissue will provide new insights to understand this metabolic disorder, but adipose tissue is not easy to sample in population-based studies. We aimed to evaluate the capacity of circulating leukocytes to reflect the adipose tissue-specific DNA methylation status of obesity susceptibility. DNA samples isolated from subcutaneous adipose tissue and circulating leukocytes were hybridized in the Infinium HumanMethylation 450 BeadChip. Data were compared between samples from obese (n = 45) and non-obese (n = 8-10) patients by Wilcoxon-rank test, unadjusted for cell type distributions. A global hypomethylation of the differentially methylated CpG sites (DMCpGs) was observed in the obese subcutaneous adipose tissue and leukocytes. The overlap analysis yielded a number of genes mapped by the common DMCpGs that were identified to reflect the obesity state in the leukocytes. Specifically, the methylation levels of FGFRL1, NCAPH2, PNKD and SMAD3 exhibited excellent and statistically significant efficiencies in the discrimination of obesity from non-obesity status (AUC > 0.80; p < 0.05) and a great correlation between both tissues. Therefore, the current study provided new and valuable DNA methylation biomarkers of obesity-related adipose tissue pathogenesis through peripheral blood analysis, an easily accessible and minimally invasive biological material instead of adipose tissue.

Genome-wide DNA methylation pattern in visceral adipose tissue differentiates insulin-resistant from insulin-sensitive obese subjects.

Elucidating the potential mechanisms involved in the detrimental effect of excess body weight on insulin action is an important priority in counteracting obesity-associated diseases. The present study aimed to disentangle the epigenetic basis of insulin resistance by performing a genome-wide epigenetic analysis in visceral adipose tissue (VAT) from morbidly obese patients depending on the insulin sensitivity evaluated by the clamp technique. The global human methylome screening performed in VAT from 7 insulin-resistant (IR) and 5 insulin-sensitive (IS) morbidly obese patients (discovery cohort) analyzed using the Infinium HumanMethylation450 BeadChip array identified 982 CpG sites able to perfectly separate the IR and IS samples. The identified sites represented 538 unique genes, 10% of which were diabetes-associated genes. The current work identified novel IR-related genes epigenetically regulated in VAT, such as COL9A1, COL11A2, CD44, MUC4, ADAM2, IGF2BP1, GATA4, TET1, ZNF714, ADCY9, TBX5, and HDACM. The gene with the largest methylation fold-change and mapped by 5 differentially methylated CpG sites located in island/shore and promoter region was ZNF714. This gene presented lower methylation levels in IR than in IS patients in association with increased transcription levels, as further reflected in a validation cohort (n = 24; 11 IR and 13 IS). This study reveals, for the first time, a potential epigenetic regulation involved in the dysregulation of VAT that could predispose patients to insulin resistance and future type 2 diabetes in morbid obesity, providing a potential therapeutic target and biomarkers for counteracting this process.

Genome-Wide DNA Methylation Analysis Identifies Novel Hypomethylated Non-Pericentromeric Genes with Potential Clinical Implications in ICF Syndrome.

INTRODUCTION AND RESULTS: Immunodeficiency, centromeric instability and facial anomalies syndrome (ICF) is a rare autosomal recessive disease, characterized by severe hypomethylation in pericentromeric regions of chromosomes (1, 16 and 9), marked immunodeficiency and facial anomalies. The majority of ICF patients present mutations in the DNMT3B gene, affecting the DNA methyltransferase activity of the protein. In the present study, we have used the Infinium 450K DNA methylation array to evaluate the methylation level of 450,000 CpGs in lymphoblastoid cell lines and untrasformed fibroblasts derived from ICF patients and healthy donors. Our results demonstrate that ICF-specific DNMT3B variants A603T/STP807ins and V699G/R54X cause global DNA hypomethylation compared to wild-type protein. We identified 181 novel differentially methylated positions (DMPs) including subtelomeric and intrachromosomic regions, outside the classical ICF-related pericentromeric hypomethylated positions. Interestingly, these sites were mainly located in intergenic regions and inside the CpG islands. Among the identified hypomethylated CpG-island associated genes, we confirmed the overexpression of three selected genes, BOLL, SYCP2 and NCRNA00221, in ICF compared to healthy controls, which are supposed to be expressed in germ line and silenced in somatic tissues. CONCLUSIONS: In conclusion, this study contributes in clarifying the direct relationship between DNA methylation defect and gene expression impairment in ICF syndrome, identifying novel direct target genes of DNMT3B. A high percentage of the DMPs are located in the subtelomeric regions, indicating a specific role of DNMT3B in methylating these chromosomal sites. Therefore, we provide further evidence that hypomethylation in specific non-pericentromeric regions of chromosomes might be involved in the molecular pathogenesis of ICF syndrome. The detection of DNA hypomethylation at BOLL, SYCP2 and NCRNA00221 may pave the way for the development of specific clinical biomarkers with the aim to facilitate the identification of ICF patients.

Pre-treatment circulating leptin/ghrelin ratio as a non-invasive marker to identify patients likely to regain the lost weight after an energy restriction treatment.

BACKGROUND: Leptin and ghrelin appear to play a role in weight regain after a successful weight loss. The pre-treatment plasma levels of leptin/ghrelin ratio (L/G) could have power to predict this clinically relevant issue in the obesity treatment. OBJECTIVE: To evaluate the ability of the L/G as a non-invasive tool for the early discrimination of obese patients who are more likely to regain weight after an energy restriction program (regainers) from those who maintain the lost weight (non-regainers). SUBJECTS AND METHODS: Fasting leptin and ghrelin levels were evaluated in 88 overweight/obese patients who followed an 8-week hypocaloric diet program and were categorized as regainers (≥10 % weight-lost regain) and non-regainers (<10 % weight-lost regain) 6 months (32 weeks) after finishing the dietary treatment. A receiver operating characteristic (ROC) curve analysis was employed to evaluate the diagnostic value of the L/G ratio and to establish a cut-off point to differentiate regainers from non-regainers. RESULTS: Regainers showed a statistically higher baseline (week 0) and after treatment (week 8) L/G ratio than non-regainers. The baseline L/G ratio was associated with an increased risk for weight regain (odds ratio 1.051; p = 0.008). Using the area under the ROC curve (AUC), the L/G ratio significantly identified female (AUC = 0.69; p = 0.040) and male regainers (AUC = 0.68; p = 0.030). The maximum combination of sensitivity and specificity was shown at the cut-off point of 26.0 for women and 9.5 for men. CONCLUSIONS: The pre-intervention fasting leptin/ghrelin ratio could be a useful non-invasive approach to personalize obesity therapy and avoid unsuccessful treatment outcomes.

Oxidative stress associated to dysfunctional adipose tissue: a potential link between obesity, type 2 diabetes mellitus and breast cancer.

Diabetes mellitus and breast cancer are two important health problems. Type 2 diabetes (T2DM) and obesity are closely linked with both being associated with breast cancer. Despite abundant epidemiological data, there is no definitive evidence regarding the mechanisms responsible for this association. The proposed mechanisms by which diabetes affects breast cancer risk and prognosis are the same as the mechanisms hypothesised for the contribution of obesity to breast cancer risk. The obesity-induced inflammation promoted by adipose tissue dysfunction is a key feature, which is thought to be an important link between obesity and cancer. Inflammation induces an increase in free radicals and subsequently promotes oxidative stress, which may create a microenvironment favourable to the tumor development in obese persons. Oxidative stress is also proposed as the link between obesity and diabetes mellitus. Therefore, obesity-related oxidative stress could be a direct cause of neoplastic transformation associated with obesity and T2DM in breast cancer cells. This review is focused on the role of obesity-related oxidative stress in the context of chronic inflammation, on the time of breast cancer onset and progression, which provide targets for preventive and therapeutic strategies in the fields of diabetes and obesity-related breast cancer.

Preliminary results of the combination of bevacizumab and weekly Paclitaxel in advanced melanoma.

BACKGROUND: Pretreated advanced melanoma is a poor prognosis scenario with few, if any, active therapeutic options. The antibody against vascular endothelial growth factor, bevacizumab, has demonstrated increased activity in combination with chemotherapy in many tumors. We intended to evaluate the activity of the combination of weekly paclitaxel and bevacizumab in previously treated metastatic melanoma. PATIENTS AND METHODS: Patients with previously treated metastatic melanoma received paclitaxel 70 mg/m(2) weekly and bevacizumab 10 mg/kg biweekly for 5 consecutive weeks every 6 weeks. RESULTS: Twelve patients were treated. Two patients (16.6%) achieved a partial response and 7 patients (58.3%) stable disease. Responses were seen in soft tissue, lung and brain metastases. Median disease-free and overall survival times were 3.7 and 7.8 months, respectively. Treatment was well tolerated. Main toxicities were grade 3 asymptomatic lymphopenia in 6 patients, grade 3 leucopenia in 2 patients, and grade 3 thrombocytopenia in 1 patient. CONCLUSIONS: Our preliminary results suggest that the combination of bevacizumab and weekly paclitaxel is active and safe in patients with metastatic melanoma, warranting further investigation.