Maternal cigarette smoking during pregnancy and offspring DNA methylation in midlife

Maternal smoking in pregnancy (MSP) has been associated with DNA methylation in specific CpG sites (CpGs) in infants and children. We investigated whether MSP, independent of own personal active smoking, was associated with midlife DNA methylation in CpGs that were previously identified in studies of MSP-DNA methylation in children. We used data on MSP collected from pregnant mothers of 89 adult women born in 1959–1964 and measured DNA methylation in blood (granulocytes) collected in 2001–2007 (mean age: 43 years). Seventeen CpGs were differentially methylated by MSP, with multiple CpGs mapping to CYP1A1, MYO1G, AHRR, and GFI1. These associations were consistent in direction with prior studies (e.g., MSP associated with more and less methylation in AHRR and CYP1A1, respectively) and, with the exception of AHRR CpGs, were not substantially altered by adjustment for active smoking. These preliminary results confirm prior prospective reports that MSP influences the offspring DNA methylation, and extends the timeframe to midlife, and suggest that these effects may persist into adulthood, independently of active smoking.     

Lower Methylation of the ANGPTL2 Gene in Leukocytes from Post-Acute Coronary Syndrome Patients

DNA methylation is believed to regulate gene expression during adulthood in response to the constant changes in environment. The methylome is therefore proposed to be a biomarker of health through age. ANGPTL2 is a circulating pro-inflammatory protein that increases with age and prematurely in patients with coronary artery diseases; integrating the methylation pattern of the promoter may help differentiate age- vs. disease-related change in its expression. We believe that in a pro-inflammatory environment, ANGPTL2 is differentially methylated, regulating ANGPTL2 expression. To test this hypothesis we investigated the changes in promoter methylation of ANGPTL2 gene in leukocytes from patients suffering from post-acute coronary syndrome (ACS). DNA was extracted from circulating leukocytes of post-ACS patients with cardiovascular risk factors and from healthy young and age-matched controls. Methylation sites (CpGs) found in the ANGPTL2 gene were targeted for specific DNA methylation quantification. The functionality of ANGPTL2 methylation was assessed by an in vitro luciferase assay. In post-ACS patients, C-reactive protein and ANGPTL2 circulating levels increased significantly when compared to healthy controls. Decreased methylation of specific CpGs were found in the promoter of ANGPTL2 and allowed to discriminate age vs. disease associated methylation. In vitro DNA methylation of specific CpG lead to inhibition of ANGPTL2 promoter activity. Reduced leukocyte DNA methylation in the promoter region of ANGPTL2 is associated with the pro-inflammatory environment that characterizes patients with post-ACS differently from age-matched healthy controls. Methylation of different CpGs in ANGPTL2 gene may prove to be a reliable biomarker of coronary disease.     

CLOCK,PER2 and BMAL1 DNA Methylation: Association with Obesity and Metabolic Syndrome Characteristics and Monounsaturated Fat Intake

The circadian clock system instructs 24-h rhythmicity on gene expression in essentially all cells, including adipocytes, and epigenetic mechanisms may participate in this regulation. The aim of this research was to investigate the influence of obesity and metabolic syndrome (MetS) features in clock gene methylation and the involvement of these epigenetic modifications in the outcomes. Sixty normal-weight, overweight and obese women followed a 16-weeks weight reduction program. DNA methylation levels at different CpG sites of CLOCK, BMAL1 and PER2 genes were analyzed by Sequenom's MassARRAY in white blood cells obtained before the treatment. Statistical differences between normal-weight and overweight?+?obese subjects were found in the methylation status of different CpG sites of CLOCK (CpGs 1, 5-6, 8 and 11-14) and, with lower statistical significance, in BMAL1 (CpGs 6-7, 8, 15 and 16-17). The methylation pattern of different CpG sites of the three genes showed significant associations with anthropometric parameters such as body mass index and adiposity, and with a MetS score. Moreover, the baseline methylation levels of CLOCK CpG 1 and PER2 CpGs 2-3 and 25 correlated with the magnitude of weight loss. Interestingly, the percentage of methylation of CLOCK CpGs 1 and 8 showed associations with the intake of monounsaturated and polyunsaturated fatty acids. This study demonstrates for the first time an association between methylation status of CpG sites located in clock genes (CLOCK, BMAL1 and PER2) with obesity, MetS and weight loss. Moreover, the methylation status of different CpG sites in CLOCK and PER2 could be used as biomarkers of weight-loss success, particularly CLOCK CPGs 5-6. (Author correspondence: garaulet@um.es)     

Epigenome-wide differential DNA methylation between HIV-infected and uninfected individuals

Epigenetic control of human immunodeficiency virus-1 (HIV-1) genes is critical for viral integration and latency. However, epigenetic changes in the HIV-1-infected host genome have not been well characterized. Here, we report the first large-scale epigenome-wide association study of DNA methylation for HIV-1 infection. We recruited HIV-infected (n = 261) and uninfected (n = 117) patients from the Veteran Aging Cohort Study (VACS) and all samples were profiled for 485,521 CpG sites in DNA extracted from the blood. After adjusting for cell type and clinical confounders, we identified 20 epigenome-wide significant CpGs for HIV-1 infection. Importantly, 2 CpGs in the promoter of the NLR family, CARD domain containing gene 5 (NLRC5), a key regulator of major histocompatibility complex class I gene expression, showed significantly lower methylation in HIV-infected subjects than in uninfected subjects (cg07839457: t = ?6.03, P_nominal = 4.96 × 10^?9; cg16411857: t = ?7.63, P_nominal = 3.07 × 10^?13). Hypomethylation of these 2 CpGs was replicated in an independent sample (GSE67705: cg07839457: t = ?4.44, P_nominal = 1.61 × 10^?5; cg16411857: t = ?5.90; P = 1.99 × 10^?8). Methylation of these 2 CpGs in NLRC5 was negatively correlated with viral load in the 2 HIV-infected samples (cg07839457: P = 1.8 × 10^?4; cg16411857: P = 0.03 in the VACS; and cg07839457: P = 0.04; cg164111857: P = 0.01 in GSE53840). Our findings demonstrate that differential DNA methylation is associated with HIV infection and suggest the involvement of a novel host gene, NLRC5, in HIV pathogenesis.     

Perinatal Nicotine Exposure Increases Angiotensin II Receptor-Mediated Vascular Contractility in Adult Offspring

Previous studies have reported that perinatal nicotine exposure causes development of hypertensive phenotype in adult offspring.

Aims

The present study was to determine whether perinatal nicotine exposure causes an epigenetic programming of vascular Angiotensin II receptors (ATRs) and their-mediated signaling pathway leading to heightened vascular contraction in adult offspring.

Main methods

Nicotine was administered to pregnant rats via subcutaneous osmotic minipumps from day 4 of gestation to day 10 after birth. The experiments were conducted at 5 months of age of male offspring.

Key Findings

Nicotine treatment enhanced Angitension II (Ang II)-induced vasoconstriction and 20-kDa myosin light chain phosphorylation (MLC₂₀-P) levels. In addition, the ratio of Ang II-induced tension/MLC-P was also significantly increased in nicotine-treated group compared with the saline group. Nicotine-mediated enhanced constrictions were not directly dependent on the changes of [Ca²⁺]_i concentrations but dependent on Ca²⁺ sensitivity. Perinatal nicotine treatment significantly enhanced vascular ATR type 1a (AT_1aR) but not AT_1bR mRNA levels in adult rat offspring, which was associated with selective decreases in DNA methylation at AT_1aR promoter. Contrast to the effect on AT_1aR, nicotine decreased the mRNA levels of vascular AT₂R gene, which was associated with selective increases in DNA methylation at AT₂R promoter.

Significance

Our results indicated that perinatal nicotine exposure caused an epigenetic programming of vascular ATRs and their-mediated signaling pathways, and suggested that differential regulation of AT₁R/AT₂R gene expression through DNA methylation mechanism may be involved in nicotine-induced heightened vasoconstriction and development of hypertensive phenotype in adulthood.     

DNA methylation pattern of CALCA in preterm neonates with bacterial sepsis as a putative epigenetic biomarker

Diagnosis of bacterial sepsis in preterm neonates can be difficult when using serum markers that rely on physiological changes because these changes may not necessarily be the result of bacterial infections alone. This retrospective investigation explores the potential use of the DNA methylation pattern of CpG sites in the promoter region of the calcitonin-related polypeptide α (CALCA) gene as an epigenetic biomarker for bacterial sepsis in preterm newborns. Four novel changes in the DNA methylation of eight CpG sites were detected in this gene and are present only in neonates with bacterial sepsis: (1) partial methylation at -769 CpG in gram-negative or gram-positive early onset sepsis (EOS) and late onset sepsis (LOS) episodes; (2) demethylation of 8 CpGs in gram-negative EOS followed by LOS (ELS) and in gram-negative EOS; (3) demethylation of 7 CpGs in gram-positive ELS and gram-positive EOS; (4) -771 C:G > T:A; 5′ de novo -778 CpG mutation on both alleles in EOS. These changes were not detected in birth weight and gestational age matched controls or in newborns with isolated infections. Our results indicate that the DNA methylation pattern of the promoter region of the CALCA gene varies in different types of bacterial preterm sepsis, thus suggesting a potential use as an epigenetic biomarker. A prospective confirmation of these results is essential.     

Circulating cell-free DNA of methylated insulin-like growth factor-binding protein 7 predicts a poor prognosis in hepatitis B virus-associated hepatocellular carcinoma after hepatectomy

The initiation and progression of hepatocellular carcinoma (HCC) is a multistage process involving a variety of changes at the gene level. Methylation of insulin-like growth factor-binding protein 7 (IGFBP7) plays a crucial role in HCC development. The main purpose of this study was to investigate the relationship between oxidative stress, DNA methyltransferases (DNMTs) expression, and IGFBP7 methylation, and to evaluate the prognostic value of serum IGFBP7 methylation status in patients with HCC after hepatectomy. We enrolled 155 patients with HCC undergoing surgical resection. The IGFBP7 methylation status, DNMTs mRNA levels and malondialdehyde (MDA), xanthine oxidase (XOD), reduced glutathione hormone (GSH), and glutathione-S-transferases (GST) levels were detected. MDA and XOD levels were significantly higher in IGFBP7 methylated group than unmethylated group, while GSH level was lower in methylated group than unmethylated group. The DNMT1 and DNMT3a mRNA levels were higher in IGFBP7 methylated group than unmethylated group. Kaplan–Meier curve analysis revealed that IGFBP7 promoter methylation was significantly correlated with overall survival (OS) (p?IGFBP7 methylation was an independent prognostic predictor for OS (p?=?.000) and early tumour recurrence (ETR) (p?=?.008) in HCC after hepatectomy. Our results indicated that IGFBP7 promoter methylation was associated with oxidative stress and DNMTs expression. Meanwhile, IGFBP7 promoter methylation was associated with OS and ETR, indicating that it might serve as a potentially independent prognostic factor in patients with HCC after hepatectomy.     

CpG dinucleotide-specific hypermethylation of the TNS3 gene promoter in human renal cell carcinoma

Tensin3 is a cytoskeletal regulatory protein that inhibits cell motility. Downregulation of the gene encoding Tensin3 (TNS3) in human renal cell carcinoma (RCC) may contribute to cancer cell metastatic behavior. We speculated that epigenetic mechanisms, e.g., gene promoter hypermethylation, might account for TNS3 downregulation. In this study, we identified and validated a TNS3 gene promoter containing a CpG island, and quantified the methylation level within this region in RCC. Using a luciferase reporter assay we demonstrated a functional minimal promoter activity for a 500-bp sequence within the TNS3 CpG island. Pyrosequencing enabled quantitative determination of DNA methylation of each CpG dinucleotide (a total of 43) in the TNS3 gene promoter. Across the entire analyzed CpG stretch, RCC DNA showed a higher methylation level than both non-tumor kidney DNA and normal control DNA. Out of all the CpGs analyzed, two CpG dinucleotides, specifically position 2 and 8, showed the most pronounced increases in methylation levels in tumor samples. Furthermore, CpG-specific higher methylation levels were correlated with lower TNS3 gene expression levels in RCC samples. In addition, pharmacological demethylation treatment of cultured kidney cells caused a 3-fold upregulation of Tensin3 expression. In conclusion, these results reveal a differential methylation pattern in the TNS3 promoter occurring in human RCC, suggesting an epigenetic mechanism for aberrant Tensin downregulation in human kidney cancer.