Xiaojing Xu

A genome-wide methylation study on obesity: Differential variability and differential methylation

Besides differential methylation, DNA methylation variation has recently been proposed and demonstrated to be a potential contributing factor to cancer risk. Here we aim to examine whether differential variability in methylation is also an important feature of obesity, a typical non-malignant common complex disease. We analyzed genome-wide methylation profiles of over 470,000 CpGs in peripheral blood samples from 48 obese and 48 lean African-American youth aged 14–20 y old. A substantial number of differentially variable CpG sites (DVCs), using statistics based on variances, as well as a substantial number of differentially methylated CpG sites (DMCs), using statistics based on means, were identified. Similar to the findings in cancers, DVCs generally exhibited an outlier structure and were more variable in cases than in controls. By randomly splitting the current sample into a discovery and validation set, we observed that both the DVCs and DMCs identified from the first set could independently predict obesity status in the second set. Furthermore, both the genes harboring DMCs and the genes harboring DVCs showed significant enrichment of genes identified by genome-wide association studies on obesity and related diseases, such as hypertension, dyslipidemia, type 2 diabetes and certain types of cancers, supporting their roles in the etiology and pathogenesis of obesity. We generalized the recent finding on methylation variability in cancer research to obesity and demonstrated that differential variability is also an important feature of obesity-related methylation changes. Future studies on the epigenetics of obesity will benefit from both statistics based on means and statistics based on variances.

DNA methylation mediates the effect of maternal smoking during pregnancy on birthweight of the offspring

Background: We examined whether the effect of maternal smoking during pregnancy on birthweight of the offspring was mediated by smoking-induced changes to DNA methylation in cord blood. Methods: First, we used cord blood of 129 Dutch children exposed to maternal smoking vs 126 unexposed to maternal and paternal smoking (53% male) participating in the GECKO Drenthe birth cohort. DNA methylation was measured using the Illumina HumanMethylation450 Beadchip. We performed an epigenome-wide association study for the association between maternal smoking and methylation followed by a mediation analysis of the top signals [false-discovery rate (FDR) < 0.05]. We adjusted both analyses for maternal age, education, pre-pregnancy BMI, offspring’s sex, gestational age and white blood cell composition. Secondly, in 175 exposed and 1248 unexposed newborns from two independent birth cohorts, we replicated and meta-analysed results of eight cytosine-phosphate-guanine (CpG) sites in the GFI1 gene, which showed the most robust mediation. Finally, we performed functional network and enrichment analysis. Results: We found 35 differentially methylated CpGs (FDR < 0.05) in newborns exposed vs unexposed to smoking, of which 23 survived Bonferroni correction (P < 1 × 10-7). These 23 CpGs mapped to eight genes: AHRR, GFI1, MYO1G, CYP1A1, NEUROG1, CNTNAP2, FRMD4A and LRP5. We observed partial confirmation as three of the eight CpGs in GFI1 replicated. These CpGs partly mediated the effect of maternal smoking on birthweight (Sobel P < 0.05) in meta-analysis of GECKO and the two replication cohorts. Differential methylation of these three GFI1 CpGs explained 12–19% of the 202 g lower birthweight in smoking mothers. Functional enrichment analysis pointed towards activation of cell-mediated immunity. Conclusions: Maternal smoking during pregnancy was associated with cord blood methylation differences. We observed a potentially mediating role of methylation in the association between maternal smoking during pregnancy and birthweight of the offspring. Functional network analysis suggested a role in activating the immune system.

A Genome-Wide Methylation Study on Essential Hypertension in Young African American Males

Objective There is emerging evidence from animal studies suggesting a key role for methylation in the pathogenesis of essential hypertension. However, to date, very few studies have investigated the role of methylation in the development of human hypertension, and none has taken a genome-wide approach. Based on the recent studies that highlight the involvement of inflammation in the development of hypertension, we hypothesize that changes in DNA methylation of leukocytes are involved in the pathogenesis of hypertension. Method & Results We conducted a genome-wide methylation analysis on 8 hypertensive cases and 8 normotensive age-matched controls aged 14–23 years and performed validation of the most significant CpG sites in 2 genes in an independent sample of 36 hypertensive cases and 60 normotensive controls aged 14–30 years. Validation of the CpG sites in the SULF1 gene was further conducted in a second replication sample of 36 hypertensive cases and 34 controls aged 15.8–40 years. A CpG site in the SULF1 gene showed higher methylation levels in cases than in healthy controls in the genome-wide step (p = 6.2×10−5), which was confirmed in the validation step (p = 0.011) for subjects ≤30 years old but was not significant for subjects of all ages combined (p = 0.095). Conclusion The identification of a difference in a blood leukocyte DNA methylation site between hypertensive cases and normotensive controls suggests that changes in DNA methylation may play an important role in the pathogenesis of hypertension. The age dependency of the effect further suggests complexity of epigenetic regulation in this age-related disease.

Adverse Childhood Experiences and Blood Pressure Trajectories From Childhood to Young Adulthood The Georgia Stress and Heart Study

Background— The purposes of this study were to assess the long-term effect of adverse childhood experiences (ACEs) on blood pressure (BP) trajectories from childhood to young adulthood and to examine whether this relation is explained by childhood socioeconomic status (SES) or risk behaviors that are associated with ACEs. Methods and Results— Systolic and diastolic BPs were measured up to 16 times (13 times on average) over a 23-year period in 213 African Americans and 181 European Americans 5 to 38 years of age. Retrospective data on traumatic experiences before 18 years of age were collected, including abuse, neglect, and household dysfunction. Individual growth curve modeling within a multilevel framework was used to examine the relation between exposure to ACEs and BP development. No main effect of ACEs on average BP levels was found. However, a significant interaction of ACE score with age3 was observed (systolic BP, P=0.033; diastolic BP, P=0.017). Subjects who experienced multiple traumatic events during childhood showed a faster rise in BP levels after 30 years of age than those without ACEs. As expected, a graded association of ACEs with childhood socioeconomic status and negative health behaviors was observed (P<0.001). The ACE–systolic BP relation was not explained by these factors, whereas the ACE–diastolic BP relation was partially mediated by illicit drug use. Conclusion— In this novel longitudinal study, we observed that participants who were exposed to multiple ACEs displayed a greater increase in BP levels in young adulthood compared with their counterparts without ACEs.

DNA Methylation of the LY86 Gene is Associated With Obesity, Insulin Resistance, and Inflammation

BACKGROUND Previous genome-wide association studies (GWAS) have identified a large number of genetic variants for obesity and its related traits, representing a group of potential key genes in the etiology of obesity. Emerging evidence suggests that epigenetics may play an important role in obesity. It has not been explored whether the GWAS-identified loci contribute to obesity through epigenetics (e.g., DNA (deoxyribonucleic acid) methylation) in addition to genetics. METHOD A multi-stage cross-sectional study was designed. We did a literature search and identified 117 genes discovered by GWAS for obesity and its related traits. Then we analyzed whether the methylation levels of these genes were also associated with obesity in two genome-wide methylation panels. We examined an initial panel of seven adolescent obese cases and seven age-matched lean controls, followed by a second panel of 48 adolescent obese cases and 48 age- and gender-matched lean controls. The validated CpG sites were further replicated in two independent replication panels of youth (46 vs. 46 and 230 cases vs. 413 controls, respectively) and a general population of youth, including 703 healthy subjects. RESULTS One CpG site in the lymphocyte antigen 86 (LY86) gene, which showed higher methylation in the obese in both the initial (p = .009) and second genome-wide DNA methylation panel (p = .008), was further validated in both replication panels (meta p = .00016). Moreover, in the general population of youth, the methylation levels of this region were significantly correlated with adiposity indices (p ≤ .02), insulin resistance (p = .001), and inflammatory markers (p < .001). CONCLUSION By focusing on recent GWAS findings in genome-wide methylation profiles, we identified a solid association between LY86 gene DNA methylation and obesity.

Genetic and environmental influences on blood pressure variability: A study in twins

Objectives: Blood pressure variability (BPV) and its reduction in response to antihypertensive treatment are predictors of clinical outcomes; however, little is known about its heritability. In this study, we examined the relative influence of genetic and environmental sources of variance of BPV and the extent to which it may depend on race or sex in young twins. Methods: Twins were enrolled from two studies. One study included 703 white twins (308 pairs and 87 singletons) aged 18–34 years, whereas another study included 242 white twins (108 pairs and 26 singletons) and 188 black twins (79 pairs and 30 singletons) aged 12–30 years. BPV was calculated from 24-h ambulatory blood pressure recording. Results: Twin modeling showed similar results in the separate analysis in both twin studies and in the meta-analysis. Familial aggregation was identified for SBP variability (SBPV) and DBP variability (DBPV) with genetic factors and common environmental factors together accounting for 18–40% and 23–31% of the total variance of SBPV and DBPV, respectively. Unique environmental factors were the largest contributor explaining up to 82–77% of the total variance of SBPV and DBPV. No sex or race difference in BPV variance components was observed. The results remained the same after adjustment for 24-h blood pressure levels. Conclusions: The variance in BPV is predominantly determined by unique environment in youth and young adults, although familial aggregation due to additive genetic and/or common environment influences was also identified explaining about 25% of the variance in BPV.

Obesity is associated with more activated neutrophils in African American male youth

Background:There is emerging evidence suggesting the role of peripheral blood leukocytes in the pathogenesis of obesity and related diseases. However, few studies have taken a genome-wide approach to investigating gene expression profiles in peripheral leukocytes between obese and lean individuals with the consideration of obesity-related shifts in leukocyte types.Method:We conducted this study in 95 African Americans (AAs) of both genders (age 14–20 years, 46 lean and 49 obese). Complete blood count with differential test (CBC) was performed in whole blood. Genome-wide gene expression analysis was obtained using the Illumina HumanHT-12 V4 Beadchip with RNA extracted from peripheral leukocytes. Out of the 95 participants, 64 had neutrophils stored. The validation study was based on real-time PCR with RNA extracted from purified neutrophils.Results:CBC test suggested that, in males, obesity was associated with increased neutrophil percentage (P=0.03). Genome-wide gene expression analysis showed that, in males, the majority of the most differentially expressed genes were related to neutrophil activation. Validation of the gene expression levels of ELANE (neutrophil elastase) and MPO (myeloperoxidase) in purified neutrophils demonstrated that the expression of these two genes—important biomarkers of neutrophils activation—were significantly elevated in obese males (P=0.01 and P=0.02, respectively).Conclusion:The identification of increased neutrophil percentage and activation in obese AA males suggests that neutrophils have an essential role in the pathogenesis of obesity-related disease. Further functional and mechanistic studies on neutrophils may contribute to the development of novel intervention strategies reducing the burden associated with obesity-related health problems.

Pulse wave velocity in elastic and muscular arteries: tracking stability and association with anthropometric and hemodynamic measurements

Pulse wave velocity (PWV) has been used as a measurement for arterial stiffness, a predictor of cardiovascular risk. Tracking describes the stability of a measurement over time. The purpose of this study was to evaluate the tracking stability of carotid-femoral (cfPWV), carotid-radial (crPWV) and carotid-distal (cdPWV) PWV in young adults and their associations with anthropometric and hemodynamic measurements. cfPWV, crPWV and cdPWV were measured by tonometric (SphygmoCor) technique in 531 subjects (aged 23.7±4.9 with 42.9% African Americans and 49.9% females). Out of these subjects, 142 subjects had all these 3 PWV measurements evaluated again during their next visit with an average follow-up time of 2 years. In the tracking analysis on the data from the 142 subjects, cfPWV displayed moderate to relatively high tracking ability (r=0.61, P<0.001), whereas crPWV and cdPWV only displayed low to moderate tracking coefficients (r=0.29 and r=0.36 respectively, P<0.001). In the association test on the data from the 531 subjects, all three PWV measurements showed significant correlations with age and obesity related measurements. cfPWV displayed stronger correlations with these parameters. In addition, all three PWVs showed significant correlations with systolic blood pressure, diastolic blood pressure, mean arterial pressure and pulse pressure with the exception that no correlation existed between crPWV and pulse pressure. The higher tracking ability of cfPWV and its higher association with obesity related measurements highlights the importance of using cfPWV compared with crPWV and cdPWV for research and clinical settings.

Phenotypic alteration of CD8+ T cells in chronic lymphocytic leukemia is associated with epigenetic reprogramming

Immunosuppression is a prevalent clinical feature in chronic lymphocytic leukemia (CLL) patients, with many patients demonstrating increased susceptibility to infections as well as increased failure of an antitumor immune response. However, much is currently not understood regarding the precise mechanisms that attribute to this immunosuppressive phenotype in CLL. To provide further clarity to this particular phenomenon, we analyzed the T-cell profile of CLL patient samples within a large cohort and observed that patients with an inverted CD4/CD8 ratio had a shorter time to first treatment as well as overall survival. These observations coincided with higher expression of the immune checkpoint receptor PD-1 in CLL patient CD8+ T cells when compared to age-matched healthy donors. Interestingly, we discovered that increased PD-1 expression in CD8+ T cells corresponds with decreased DNA methylation levels in a distal upstream locus of the PD-1 gene PDCD1. Further analysis using luciferase reporter assays suggests that the identified PDCD1 distal upstream region acts as an enhancer for PDCD1 transcription and this region becomes demethylated during activation of naïve CD8+ T cells by anti-CD3/anti-CD28 antibodies and IL2. Finally, we conducted a genome-wide DNA methylation analysis comparing CD8+ T cells from CLL patients against healthy donors and identified additional differentially methylated genes with known immune regulatory functions including CCR6 and KLRG1. Taken together, our findings reveal the occurrence of epigenetic reprogramming taking place within CLL patient CD8+ T cells and highlight the potential mechanism of how immunosuppression is accomplished in CLL.

Specific Genetic Influences on Nighttime Blood Pressure

OBJECTIVES Nighttime blood pressure (BP) has been shown to be superior to daytime BP in predicting hypertension related target organ damage and cardiac mortality. In our Georgia Cardiovascular Twin Study, we showed that apart from the genes that also influence daytime BP, specific genetic determinants explained 44% and 67% of the nighttime systolic BP (SBP) and diastolic BP (DBP) heritabilities, respectively. Here, we determined whether these results could be confirmed in a much larger twin cohort of young adults with 24-hour ambulatory BP measurements. METHODS Ambulatory BP was available in 703 white twins (308 pairs and 87 singletons, aged 18-34 years, 50% males) from the Prenatal Programming Twin Study. A bivariate quantitative genetic twin model was used to analyze daytime and nighttime BP. We conducted a meta-analysis to compare and integrate results from the 2 twin cohorts. RESULTS Model fitting showed no sex differences for any of the measures. Heritabilities were 0.60 and 0.51 for SBP and 0.54 and 0.46 for DBP at daytime and nighttime. The specific heritability due to novel genetic effects emerging during the nighttime was 0.21 for SBP and 0.26 for DBP, which comprised 41% and 57% of the total nighttime heritability for SBP and DBP, respectively. Meta-analysis confirmed absence of cohort differences with very similar combined results. CONCLUSIONS In addition to genes that influence both daytime and nighttime BP, a large part of the heritability is explained by genes that specifically influence BP at night.