Alcohol consumption, depression, overweight and cortisol levels as determining factors for NR3C1 gene methylation

The NR3C1 glucocorticoid receptor (GR) gene is a component of the stress response system, which can be regulated by epigenetic mechanisms. NR3C1 methylation has been associated with trauma and mental issues, including depression, post-traumatic stress, anxiety, and personality disorders. Previous studies have reported that stressful events are involved in NR3C1 gene methylation, suggesting that its regulation under environmental effects is complex. The present study aimed to analyze associations involving stressors such as socioeconomic status, health conditions, and lifestyle in relation to NR3C1 methylation in adults. This study included 386 individual users of the Brazilian Public Unified Health System (SUS), and evaluated socioeconomic and health conditions, body mass index, cortisol levels, and lifestyle. Data were correlated with NR3C1 methylation, determined using DNA pyrosequencing. The results showed that alcohol consumption, overweight, and high cortisol levels were related to NR3C1 demethylation, while depression was related to its methylation. Habits, lifestyle, and health status may influence NR3C1 gene regulation via methylation, revealing the complexity of environmental impacts on NR3C1 methylation.

Nonsyndromic orofacial clefts in Chile: LINE-1 methylation and MTHFR variants

Aim: To evaluate the risk of nonsyndromic orofacial clefts (NSOFCs) associated with LINE-1 methylation, as a marker of global DNA methylation, and the effect of MTHFR functional variants on this variable. Patients & methods: LINE-1 methylation was evaluated by bisulfite modification coupled to DNA pyrosequencing in 95 NSOFC cases and 95 controls. In these subjects, MTHFR genotypes for variants c.C677T (rs1801133) and c.A1298C (rs1801131) were obtained. Results: Middle levels (second tertile) of LINE-1 methylation increase the risk of NSOFCs. In addition, LINE-1 methylation depends on c.A1298C genotypes in controls but not in cases. Conclusion: A nonlinear association between global DNA methylation and NSOFCs was detected in this Chilean population, which appears to be influenced by MTHFR functional variants.

Activation of SIRT6 by DNA hypomethylating agents and clinical consequences on combination therapy in leukemia

The FDA-approved DNA hypomethylating agents (DHAs) like 5-azacytidine (5AC) and decitabine (DAC) demonstrate efficacy in the treatment of hematologic malignancies. Despite previous reports that showed histone acetylation changes upon using these agents, the exact mechanism underpinning these changes is unknown. In this study, we investigated the relative potency of the nucleoside analogs and non-nucleoside analogs DHAs on DNA methylation reversal using DNA pyrosequencing. Additionally, we screened their effect on the enzymatic activity of the histone deacetylase sirtuin family (SIRT1, SIRT2, SIRT3, SIRT5 and SIRT6) using both recombinant enzymes and nuclear lysates from leukemia cells. The nucleoside analogs (DAC, 5AC and zebularine) were the most potent DHAs and increased the enzymatic activity of SIRT6 without showing any significant increase in other sirtuin isoforms. ChIP-Seq analysis of bone marrow cells derived from six acute myeloid leukemia (AML) patients and treated with the nucleoside analog DAC induced genome-wide acetylation changes in H3K9, the physiological substrate for SIRT6. Data pooling from the six patients showed significant acetylation changes in 187 gene loci at different chromosomal regions including promoters, coding exons, introns and distal intergenic regions. Signaling pathway analysis showed that H3K9 acetylation changes are linked to AML-relevant signaling pathways like EGF/EGFR and Wnt/Hedgehog/Notch. To our knowledge, this is the first report to identify the nucleoside analogs DHAs as activators of SIRT6. Our findings provide a rationale against the combination of the nucleoside analogs DHAs with SIRT6 inhibitors or chemotherapeutic agents in AML due to the role of SIRT6 in maintaining genome integrity and DNA repair.

Pyrosequencing on a glass surface

We demonstrated DNA pyrosequencing at the plain hydrophobically coated surface of a microscope glass cover slip using open-surface microfluidics.

Vascular Endothelial Growth Factor Gene Polymorphisms and Risk of Cervical Intraepithelial Neoplasia

Objective:To evaluate the association between 3 vascular endothelial growth factor (VEGF) gene polymorphisms and susceptibility of cervical intraepithelial neoplasia (CIN).Materials and Methods:This prospectively collected case-control study investigates three common VEGF gene polymorphisms (ie, VEGF −460 [rs833061], VEGF +405 [rs2010963], and VEGF +936 [rs3025039]) in 203 women with CIN and 209 healthy women by DNA pyrosequencing. Associations between polymorphisms and CIN risk are evaluated with univariate and multivariable models and haplotype analysis.Results:In a multivariable regression model, the variant VEGF +405C allele was associated (odds ratio [OR], 2.5; 95% confidence interval [CI], 1.2-5.1], P = 0.02) with increased susceptibility of CIN independent of number of sexual partners (OR, 2.2; 95% CI, 1.1-4.6; P = 0.03) and smoking (OR, 3.3; 95% CI, 1.6-6.6; P = 0.001). The haplotype VEGF −460C - +405C - +936C was associated with an OR of 5.2 (95% CI, 1.2-52.7) for the susceptibility of CIN.Conclusions:The presence of the variant VEGF +405C allele and the haplotype VEGF −460C - +405C - +936C are independently associated with higher susceptibility of CIN.