Alteration of oncogenic IGF-II gene methylation status associates with hepatocyte malignant transformation

BackgroundOncogenic insulin-like growth factor-II (IGF-II) is overexpressed in hepatocellular carcinoma (HCC). The present study aimed to analyze the dynamic alteration of IGF-II CpG site methylation status and its molecular mechanism in HCC progression.MethodsIGF-II alterations were observed in rat hepatocarcinogenesis models induced by 2-acetylaminofluorene. Liver IGF-II expression was compared by immunohistochemistry or tissue IGF-II specific concentration (nmol/mg protein). Status of human IGF-II promoter 3 (P3) or rat IGF-II P2 CpG site methylation was amplified by methylation-specific polymerase chain reaction (MSP). Serum IGF-II levels were quantitatively detected by an enzyme-linked immunosorbent assay.ResultsThe levels of hepatic IGF-II expression were significantly elevated in the HCC group (P < 0.001). The unmethylation rate of IGF-II P3 CpG sites was 100% in the HCC-, 52.5% in the paracancerous-, and none (0%) in the distal noncancerous-tissues. Abnormal IGF-II expression was related to differentiation degree, tumor invasion, and positive HBV-DNA (all P < 0.001), with a negative correlation between P3 methylation degree and IGF-II expression. There was a positive correlation between liver IGF-II specific concentration and circulating IGF-II level (r = 0.97, P < 0.001). Significantly negative correlation was found between IGF-II P2 CpG site methylation and circulating IGF-II (r_s = ?0.89, P < 0.001) or liver IGF-II level (r_s = ?0.84, P < 0.001).ConclusionsThe increase of serum IGF-II and the alteration of oncogenic gene IGF-II methylation may be biomarkers for HCC diagnosis and DNA methylation may be the therapeutic target of HCC.     

α-Adducin gene promoter DNA methylation and the risk of essential hypertension

This study was conducted to test the association between promoter DNA methylation of α-Adducin (ADD1) gene and the risk of essential hypertension (EH). A total of 150 EH patients and 100 aged- and gender-matched controls were investigated. DNA methylation levels of five cytosine-phosphate-guanine (CpG) dinucleotides on ADD1 promoter were measured employing bisulfite pyrosequencing technology. Our results showed that females have a higher ADD1 DNA methylation than males and a significantly lower CpG1 methylation level is associated with increased risk of EH among them. As for males, a significant association between lower CpG2-5 methylation levels and increased risk of EH was shown. In addition, CpG2-5 methylation was found to be a highly significant predictor for EH among males. In females, CpG1 methylation was considered a predictor of hypertension. No significant correlations were found with biochemical measures, apart from the concentration of aspartate aminotransferase which was inversely correlated with ADD1 CpG2-5 methylation levels among female controls (r = ?0.703). These findings highlight that ADD1 methylation may have a contributing role in the pathogenesis of EH with varying implications for both genders.     

An exploratory study of long‐term health outcomes following an in‐patient multidisciplinary program for people with fibromyalgia syndrome

Objective: To examine the health status of people with fibromyalgia syndrome approximately 10 years after an intensive rehabilitation intervention to identify biopsychosocial factors for further research. Methods: Baseline data, collected upon admission to the rehabilitation intervention was compared to follow‐up data collected by telephone interview. Data was evaluated for differences and relationships using the appropriate parametric or non‐parametric tests. Results: The 29 participants were interviewed an average of 9.4 years after their admissions. All participants reported the persistence of fibromyalgia and use of related medication. Differences between baseline and follow‐up were: increased paid employment (P < 0.001), social networks (P < 0.05) and decreased stress levels (P < 0.05). Correlations with paid employment were: younger age (r_s = –0.66, P < 0.01); larger social networks (r_s = –0.40, P < 0.05) and transformation rehabilitation intervention experience (r_s = .46, P < 0.05). Follow‐up stress and sleep status were also related (r_s = 0.46, P < 0.05). Conclusion: Fibromyalgia symptoms and medication use persist over time. The wider issues concerning social integration and participation appear to be worthy of further investigation.     

A new monocyte epigenetic clock reveals nonlinear effects of alcohol consumption on biological aging in three independent cohorts (N = 2242)

Background

Assessing the effect of alcohol consumption on biological age is essential for understanding alcohol use-related comorbidities and mortality. Previously developed epigenetic clocks are mainly based on DNA methylation in heterogeneous cell types, which provide limited knowledge on the impacts of alcohol consumption at the individual cellular level. Evidence shows that monocytes play an important role in both alcohol-induced pathophysiology and the aging process. In this study, we developed a novel monocyte-based DNA methylation clock (MonoDNAmAge) to assess the impact of alcohol consumption on monocyte age.

Methods

A machine learning method was applied to select a set of chronological age-associated DNA methylation CpG sites from 1202 monocyte methylomes. Pearson correlation was tested between MonoDNAmAge and chronological age in three independent cohorts (N_total = 2242). Using the MonoDNAmAge clock and four established clocks (i.e., HorvathDNAmAge, HannumDNAmAge, PhenoDNAmAge, GrimDNAmAge), we then evaluated the effect of alcohol consumption on epigenetic aging in the three cohorts [i.e., Yale Stress Center Community Study (YSCCS), Veteran Aging Cohort Study (VACS), Women's Interagency HIV Study (WIHS)] using linear and quadratic models.

Results

The MonoDNAmAge, comprised of 186 CpG sites, was moderately to strongly correlated with chronological age in the three cohorts (r = 0.90, p = 3.12E−181 in YSCCS; r = 0.54, p = 1.75E−96 in VACS; r = 0.66, p = 1.50E−60 in WIHS). More importantly, we found a nonlinear association between MonoDNAmAge and alcohol consumption (p_model = 4.55E−08, $p_{x^2}$ = 7.80E−08 in YSCCS; p_model = 1.85E−02, $p_{x^2}$ = 3.46E−02 in VACS). Heavy alcohol consumption increased EAA_MonoDNAmAge up to 1.60 years while light alcohol consumption decreased EAA_MonoDNAmAge up to 2.66 years. These results were corroborated by the four established epigenetic clocks (i.e., HorvathDNAmAge, HannumDNAmAge, PhenoDNAmAge, GrimDNAmAge).

Conclusions

The results suggest a nonlinear relationship between alcohol consumption and its effects on epigenetic age. Considering adverse effects of alcohol consumption on health, nonlinear effects of alcohol use should be interpreted with caution. The findings, for the first time, highlight the complex effects of alcohol consumption on biological aging.     

DNA methylation as a marker for the past and future

Aberrant methylation of CpG islands in promoter regions can permanently inactivate tumor-suppressor genes, as mutations and chromosomal abnormalities do. In gastric cancers, CDKN2A, CDH1, and MLH1 are inactivated more frequently by aberrant methylation than by mutations, and novel tumor-suppressor genes inactivated by promoter methylation are being identified. We recently found that Helicobacter pylori (HP), a potent gastric carcinogen, induces aberrant methylation in gastric mucosae. When a panel of CpG islands was examined, some CpG islands were consistently methylated in gastric mucosae of individuals with HP infection, while others were resistant. The amount of methylated DNA molecules in the gastric mucosae (methylation level) fluctuated while active HP infection was present, but decreased after it was no longer present. Among individuals without active HP infection, methylation levels in the gastric mucosae were higher in individuals with gastric cancers than in those without. DNA methylation is emerging as a promising marker for past exposure to carcinogens and future risk of cancers.     

Associations between depressive symptoms and insulin resistance: The Hoorn Study

Aims/hypothesis The association between depression and insulin resistance has been investigated in only a few studies, with contradictory results reported. The aim of this study was to determine whether the association between symptoms of depression and insulin resistance varies across glucose tolerance status and between men and women.Subjects and methods Cross-sectional data from a population-based cohort study in Hoorn, a medium-sized town in the Netherlands, were analysed. The study sample consisted of 541 men and women aged 55–75 years, of whom 260 had NGT, 164 had IGT and 117 had established type 2 diabetes mellitus. Main outcome measures were insulin resistance defined by the homeostasis model assessment for insulin resistance (HOMA-IR) and symptoms of depression using the Centre for Epidemiologic Studies Depression Scale (CES-D).Results In the total sample, we found a weak positive correlation between the depressive symptoms CED-D scores and HOMA-IR scores (r _s = 0.156, p < 0.001). Even weaker associations were found in subjects with NGT (r _s = 0.041, p=0.509), in subjects with IGT (r _s = 0.112, p = 0.160) and in subjects with type 2 diabetes (r _s = 0.007, p = 0.942). The association between depressive symptoms and insulin resistance was similar for men and women.Conclusions/interpretation We found only weak associations between depressive symptoms and insulin resistance, which did not differ among different glucose metabolism subgroups or between men and women.     

The relationships of concentrations of insulin,intact proinsulin and 32–33 split proinsulin with cardiovascular risk factors in Type 2 (non-insulin-dependent) diabetic subjects

Summary Standard radioimmunoassay for insulin may substantially overestimate levels of insulin because of cross-reaction with other insulin-like molecules. We have measured concentrations of insulin, intact proinsulin and 32–33 split proinsulin using two-site monoclonal antibody based immunoradiometric assays, and of insulin by a standard radioimmunoassay (immunoreactive insulin) in 51 Type 2 (noninsulin-dependent) diabetic subjects in the fasting state. The relationships of these concentrations were sought with those of total cholesterol, high density lipoprotein cholesterol, low density lipoprotein cholesterol, triglyceride, plasminogen activator inhibitor, blood pressure, and indices of body fat distribution. Significant relationships were apparent between concentrations of immunoreactive insulin as measured by standard radioimmunoassay and triglyceride (r _s=0.42, p<0.001), total cholesterol (r _s=0.25, p=0.038), high density lipoprotein cholesterol (r _s=–0.30, p=0.018) and body mass index (r _s=0.30, p=0.017), but only the relationships with triglyceride (r _s=0.36, p=0.006) and body mass index (r _s=0.26, p=0.034) remained significant when concentrations of immunoradiometrically measured insulin were employed. Concentrations of 32—33 split proinsulin, which comprises the major insulin-like molecule in these subjects, correlated positively with triglyceride (r _s=0.33, p=0.009), total cholesterol (r _s=0.23, p=0.050), and plasminogen activator inhibitor (r _s=0.26, p=0.049), and negatively with high density lipoprotein cholesterol (r _s=–0.29, p=0.021). Concentrations of immunoreactive insulin and immunoradiometric assay insulin showed significant positive correlaion with both systolic (r _s=0.24, p=0.044 and r _s=0.29, p=0.020 respectively), and diastolic blood pressure (r _s=0.48, p<0.001 and n=0.42, p=0.001 respectively), while those of intact proinsulin and 32–33 split proinsulin correlated only with diastolic blood pressure (r _s=0.33, p=0.009 and r _s=0.31, p=0.014 respectively). Using multiple regression analysis, and including age, sex, race and body mass index in the analyses, concentrations of intact proinsulin and 32–33 split proinsulin, but not immunoradiometric assay insulin, were significantly related to diastolic blood pressure. When all three molecules were incorporated into a single model, only 32–33 split proinsulin was related to diastolic blood pressure (F-change=6.91, [5,43 degrees of freedom]; p=0.012). Thus, high concentrations of insulin-like molecules are associated with changes in recognised cardiovascular risk factor in patients with Type 2 (non-insulin-dependent) diabetes mellitus.     

PTEN hypermethylation profiles of Chinese Kazakh patients with esophageal squamous cell carcinoma

Aberrant DNA methylation of promoter region CpG islands may serve as an alternative mechanism to genetic defects in the inactivation of tumor suppressor genes (TSGs) in human malignancies. The aim of this study was to examine the promoter methylation status of the PTEN TSG and its association with esophageal squamous cell carcinoma (ESCC) carcinogenesis in a Chinese Kazakh population, which is known to have a relatively high ESCC incidence and mortality. The methylation status of the PTEN promoter region was determined in patients with ESCC (n = 95) and healthy individuals (n = 65) using highly sensitive Sequenom Epityper assays. The methylation level of the PTEN gene was significantly higher in patients with ESCC than in healthy controls. The median methylation level was 10.0% (interquartile range [IQR]: 7.0–11.0%) in patients with ESCC and 6.0% in controls (IQR: 4.0–9.0%; P = 0.001). PTEN methylation levels were higher in male patients with ESCC than in male controls, whereas a trend toward significance was observed between female patients with ESCC and female controls (P = 0.005 and P = 0.086, respectively). The PTEN methylation level was associated with histopathological grade and lymph node metastasis in patients with ESCC (P = 0.002 and P = 0.009, respectively). To our knowledge, this is the first report to show the presence of PTEN promoter CpG hypermethylation in ESCC and its association with tumor metastasis.     

Elevated levels of DNA methylation at the OPRM1 promoter region in men with opioid use disorder

Background: The mu-opioid receptor, encoded by mu-opioid receptor gene (OPRM1), has an important role in the development of addiction to opioids. Its aberrant reduction on the cell membrane is responsible, at least in part, for tolerance and physical dependence. Objectives: The present study was designed to identify the relationship between opium consumption and epigenetic mechanisms involved in opium addiction. Methods: Genomic DNA was extracted from the peripheral blood of 66 men with opium use disorder and 57 healthy men as a control group. Genomic DNAs were treated with sodium bisulfite to convert the un-methylated cytosine to uracil, while methylated cytosine remained unaffected. Nested methylation-specific PCR (MSP) was used for analyses of region 1 (R1) and region 2 (R2) of the OPRM1 promoter DNA methylation. Results: All participants were 19–56 years old, and there was no significant difference in the mean age of both groups (P = 0.082). After Bonferroni correction, results showed that the DNA methylation status significantly increased the risk of opium addiction in the R2 region compared with un-methylation status (OR = 3.80, 95%CI = 1.77–8.17, P = 0.001). However, we found no significant difference in the R1 region DNA methylation between case and control groups (21.2% and 21.1%, respectively) (P = 1). Conclusion: Our findings demonstrated DNA hypermethylation of the R2 region of the OPRM1 promoter in leukocytes of opium use disorder. In peripheral tissues such as blood, changes of epigenetic endpoints with substance use can be considered as potentially clinically useful biomarkers in identifying individuals who may warrant further diagnostic assessment of a substance use disorder.     

The utility of patient-completed and partner-completed Epworth Sleepiness Scale scores in the evaluation of obstructive sleep apnea

Purpose

Excessive daytime sleepiness in obstructive sleep apnea (OSA) is often rated differently by patients and their partners. This cross-sectional study compared the utility of patient-completed and partner-completed Epworth Sleepiness Scale (ESS) scores in the evaluation of suspected OSA.

Methods

Eighty-five patient-partner pairs were enrolled, and 75 patients completed diagnostic sleep studies. The individual and combined utilities of patient-completed and partner-completed ESS scores in identifying OSA and predicting various sleep study-derived indicators of disease severity were determined.

Results

Mean partner-completed ESS scores were higher than patient-completed ESS scores (12.3 ± 4.2 vs. 9.4 ± 4.8, p < 0.0001); Bland-Altman plot showed significant bias (partner-completed ESS scores 33.5 % higher, SD ±55.2 %). Partner-completed and combined (but not patient-completed) ESS scores correlated weakly with the apnea-hypopnea index (AHI; partner-completed ESS score r _s  = 0.25, p = 0.029; combined ESS score r _s  = 0.29, p = 0.013) and oxygen desaturation index (partner-completed ESS score r _s  = 0.26, p = 0.025; combined ESS score r _s  = 0.23, p = 0.047). None of the ESS scores correlated with body mass index, arousal index, or other parameters of nocturnal oxygen desaturation. In OSA (AHI > 15/h) detection, partner-completed ESS scores had greater sensitivity than patient-completed ESS scores (76.9 vs. 46.2 %) but poorer specificity (39.1 vs. 65.2 %); sensitivity was greatest (82.7 %) when either patient-completed or partner-completed ESS score was 10 or higher, and specificity was greatest (80.8 %) when both scores were 10 or higher.

Conclusions

Neither patient-completed nor partner-completed ESS scores by themselves have great utility in identifying OSA or predicting its severity. However, taking both scores into consideration together improves the sensitivity and specificity of the screening process.

     

Determinants of the incidence and severity of ventricular arrhythmias in aortic valve disease

The incidence of ventricular arrhythmias in patients with aortic valve disease was investigated. Twenty-four-hour ambulatory electrocardiographic recordings were obtained in 93 patients without coronary artery disease (aortic stenosis [AS], n = 38; combined AS and aortic regurgitation [AR], n = 27; and AR only, n = 28). The arrhythmias were compared with the hemodynamic findings of cardiac catheterization. Ventricular premature beats (VPB) were noted in 78 patients (84%). They were rare (< 100 VPB/22 hours) in 40 patients (43%), moderately frequent (101 to 1,000 VPB/22 hours) in 23 patients (25%), and frequent (> 1,000 VPB/22 hours) in 15 patients (16%). Multiformity was found in 47 (51%), paired VPB in 32 (34%), and ventricular tachycardia in 17 (18%) of the 93 patients studied. The occurrence of ventricular arrhythmia was not related to the type of valve lesion, to the transvalvular gradient in patients with AS, or to the degree of regurgitation in patients with AR. In contrast, the grade of arrhythmia showed a negative correlation with left ventricular ejection fraction (AS, r_s = ?0.58; AS and AR, r_s = ?0.67; AR, r_s = ?0.78; all p < 0.001) and a positive correlation with peak systolic left ventricular wall stress (AS, r_s = 0.56; AS and AR, r_s = 0.56; AR, r_s = 0.57; all p < 0.001). The frequency of VPB also showed a negative correlation with left ventricular ejection fraction (AS, r_s = ?0.63; AS and AR, r_s = ?0.65; AR, r_s = ?0.71; all p < 0.001).This study indicates that ventricular arrhythmias are present in a large number of patients with aortic valve disease. The severity of arrhythmias is strongly influenced by myocardial performance. Thus, severe arrhythmias are frequently a sign of impaired left ventricular function.     

Cannabis use by women during pregnancy does not influence infant DNA methylation of the dopamine receptor DRD4

Background: Maternal cannabis use in pregnancy is linked with long-term adverse behavioral outcomes in offspring. Epigenetic processes established in utero that affect dopaminergic (reward) signaling may mediate risks. Associations between cannabis use and offspring DNA methylation have not been investigated; however, maternal tobacco smoking in pregnancy is associated with distinct patterns of DNA methylation at birth and beyond. Objectives: To determine whether maternal cannabis use is associated with methylation of the dopamine receptor gene DRD4 promoter in infants. Methods: Mothers in the Triple B study provided detailed information on drug use in each trimester of pregnancy. Buccal swabs were collected from neonates at 8 weeks (n = 804, 51.7% male, and 48.3% female). DRD4 promoter DNA methylation was measured using SEQUENOM MassARRAY. Results: Fifty-seven of the women in the study reported drug use during pregnancy, of whom 44 used cannabis. Of 19 cytosine-phosphate-guanine dinucleotides (CpG) units tested in DRD4, gestational cannabis use was associated with offspring methylation at 1 CpG unit in multivariate models (β + 1.48, CI: 0.02 to 2.93, and p = 0.047). At another site there was weak evidence that both cannabis and other drug use were independently associated with increased methylation, while the association with tobacco was in the reverse direction (cannabis use β + 0.67, CI: ?0.12 to 1.46, and p = 0.09; other drug use β + 1.11, CI: 0.17 to 2.05, and p = 0.02; tobacco use β ?0.41, CI: ?0.85 to 0.03, and p = 0.07). None of the associations would remain significant after correction for multiple testing. Conclusion: There is no strong evidence that maternal cannabis use in pregnancy is associated with offspring DRD4 methylation.